A limited number of antibody specificities mediate broad and potent serum neutralization in selected HIV-1 infected individuals

Abstract

A protective vaccine against HIV-1 will likely require the elicitation of a broadly neutralizing antibody (bNAb) response. Although the development of an immunogen that elicits such antibodies remains elusive, a proportion of HIV-1 infected individuals evolve broadly neutralizing serum responses over time, demonstrating that the human immune system can recognize and generate NAbs to conserved epitopes on the virus. Understanding the specificities that mediate broad neutralization will provide insight into which epitopes should be targeted for immunogen design and aid in the isolation of broadly neutralizing monoclonal antibodies from these donors. Here, we have used a number of new and established technologies to map the bNAb specificities in the sera of 19 donors who exhibit among the most potent cross-clade serum neutralizing activities observed to date. The results suggest that broad and potent serum neutralization arises in most donors through a limited number of specificities (1-2 per donor). The major targets recognized are an epitope defined by the bNAbs PG9 and PG16 that is associated with conserved regions of the V1, V2 and V3 loops, an epitope overlapping the CD4 binding site and possibly the coreceptor binding site, an epitope sensitive to a loss of the glycan at N332 and distinct from that recognized by the bNAb 2G12 and an epitope sensitive to an I165A substitution. In approximately half of the donors, key N-linked glycans were critical for expression of the epitopes recognized by the bNAb specificities in the sera.

Figure 1. Serum adsorptions with recombinant Env proteins.

Sera were adsorbed with YU2 gp120 or gp140-coupled beads or blank control beads and then tested for neutralizing activity against a cross-clade pseudovirus panel using TZM-bl target cells. A) Serum neutralizing activity depleted on YU2 gp120. B) Serum neutralizing activity depleted on YU2 gp140. Percent neutralizing activity depleted on YU2 gp120 or YU2 gp140 was calculated using the equation = (1−(IC_{50 blank beads}/IC_{50 antigen-coated beads}))*100. Boxes are color coded as follows: Gray, 0–45%, yellow, 45–65%; orange, 65–85%; red, 85–100%. C) Serum neutralizing activity against the selected cross-clade pseudovirus panel. Serum IC₅₀ titers are color coded as follows: gray squares, 1∶300>IC50≥1∶100, yellow squares, 1∶500>IC50≥1∶300; orange squares, 1∶1500>IC50≥1∶500, red squares, IC50≥1∶1500.

Figure 2. Contribution of MPER-directed NAbs to broad serum neutralization.

A) Sera were tested for neutralizing activity against a chimeric HIV-2 virus containing the complete MPER region of gp41 . Serum IC₅₀ titers are expressed as 1/serum dilution. Boxes are color coded as follows: white squares, IC₅₀≤1∶80; green squares, 1∶200>IC₅₀>1∶80, yellow squares, 1∶500>IC₅₀>1∶200. The IC₅₀s of anti-MPER NAbs 2F5, 4E10, Z13e1 were also determined, expressed in µg/ml. The anti-Dengue antibody Den3 is included as a negative control. B) Serum neutralizing activity after adsorption with MPER peptide-coated beads. Sera were adsorbed with full-length MPER peptide-coated beads or blank control beads and then tested for neutralizing activity against a cross-clade pseudovirus panel, as described previously . Percent serum neutralizing activity depleted on the MPER peptide was calculated using the equation = (1−(IC_{50 blank beads}/IC_{50 MPER beads}))*100. C) Neutralizing activity of serum antibodies eluted from the MPER-coated beads. Functional Abs were eluted from the MPER-coupled beads by exposing the beads to a series of increasingly acidic conditions as described . ELISA assays were used to determine the concentration of total IgG in the eluted fraction. Boxes are color coded as follows: white squares, IC₅₀>20 µg/ml; green squares, 20≥IC₅₀>5 µg/ml, yellow squares, 5>IC₅₀>1 µg/ml.

Figure 3. Analysis of CD4bs and CRbs-directed neutralizing activity.

A) Sera were tested for neutralizing activity after adsorption with YU2 gp140-coupled beads in the presence or absence of saturating concentrations of mAb b6 or blank control beads. Percent gp140-directed neutralization to the CD4bs or CRbs was calculated using the equation = [(% neutralization absorbed by gp140 beads−% neutralization absorbed by gp140 beads in the presence of excess b6)/% neutralization absorbed by gp140 beads] *100. B) Sera were tested for neutralizing activity after adsorption with YU2 gp120 D368R coated beads or blank control beads. Percent gp120-directed neutralization sensitive to the D368R mutation was calculated using the equation = [(% neutralization absorbed by gp120 beads−% neutralization absorbed by gp120 D368R beads)/% neutralization absorbed by gp120 beads] *100. Boxes are color coded as follows: Gray, 0–45%, yellow, 45–65%; orange, 65–85%; red, 85–100%.

Figure 4. Effects of single amino acid substitutions on serum neutralizing activity against JR-CSF.

Sera were tested for neutralizing activity against 97 JR-CSF pseudoviruses incorporating single amino acid substitutions. Percent serum neutralizing activity sensitive to the indicated substitution was calculated using the equation = (1−(IC_{50 variant}/IC_{50 WT}))*100. Boxes are color coded as follows: yellow, 20–50%; orange, 50–80%; red, 80–100%. The first and second rows designate the gp120 domain and the single amino acid substitution, respectively. JR-CSF pseudovirus variants that were similarly sensitive as wild type JR-CSF to all of the sera or variants that were globally sensitive to serum neutralization are not shown but are listed in Table S1.

Figure 5. Effects of single amino acid substitutions on broad serum neutralizing activity.

A) Sera were tested for neutralizing activity against a cross-clade pseudovirus panel incorporating single amino acid substitutions using a single round of replication pseudovirus assay and TZM-bl target cells, as described previously . Percent serum neutralizing activity sensitive to the indicated substitution was calculated using the equation = (1−(IC_{50 variant}/IC_{50 WT}))*100. Boxes are color coded as follows: green, 0–20%; yellow, 20–40%; orange, 40–80%; red, 80–100%. (—) indicates that the isolate was neutralized by the serum at <1∶100 dilution. B) Serum neutralizing activity against the 5-pseudovirus panel. Serum IC₅₀ titers are color coded as follows: white squares, IC50≤1∶100; green squares, 1∶300>IC50≥1∶100, yellow squares, 1∶500>IC50≥1∶300; orange squares, 1∶1500>IC50≥1∶500, red squares, IC50≥1∶1500.

Figure 6. Analysis of glycan-specific activity in sera using TM-Pst1 coated beads.

Sera were tested for neutralizing activity after adsorption with TM-Pst1-coupled beads or blank control beads. Neutralizing activity was assessed against a cross-clade pseudovirus panel using a single round of replication pseudovirus assay using TZM-bl target cells, as described . Percent neutralizing activity cross-reactive with TM-Pst1 was calculated using the equation = (1−(IC_{50 blank bead-adsorbed serum}/IC_{50 Pst1-adsorbed serum}))*100.

Figure 7. Serum neutralization activity against kifunensine-treated JR-CSF pseudoviruses.

Sera were tested for neutralizing activity against JR-CSF pseudoviruses produced in the presence or absence of the glycosidase inhibitor kifunensine. Kifunensine-treated pseudoviruses were produced by treating 293T cells with 25 µM kifunensine on the day of transfection. Neutralizing activity was assessed using a single round of replication pseudovirus assay and TZM-bl target cells, as described previously .

Figure 8. Effect of kifunensine treatment on broad serum neutralization.

Sera were tested for neutralizing activity against a cross-clade panel of pseudoviruses produced in the presence or absence of the glycosidase inhibitor kifunensine. Kifunensine-treated pseudoviruses were produced by treating 293T cells with 25 µM kifunensine on the day of transfection. Neutralizing activity was assessed using a single round of replication pseudovirus assay and TZM-bl target cells, as described previously . Percent neutralizing activity sensitive to kifunensine treatment was calculated using the equation = (1−(IC_{50 kifunensine treated virus}/IC_{50 WT virus}))*100. Boxes are color coded as follows: green, 0–30%; yellow, 30–60%; orange, 60–90%; red, 90–100%.

Figure 9. Summary of the predominant NAb specificities that mediate serum neutralization breath and potency across all donors.

Percentages were calculated using only the predominant NAb specificity identified in each donor. The “unknown” category includes donors for which no predominant specificity was identified.