Signature biochemical properties of broadly cross-reactive HIV-1 neutralizing antibodies in human plasma

Abstract

The common properties of broadly cross-reactive HIV-1 neutralization antibodies found in certain HIV-1-infected individuals holds significant value for understanding natural and vaccine-mediated anti-HIV immunity. Recent efforts have addressed this question by deriving neutralizing monoclonal anti-envelope antibodies from memory B cell pools of selected subjects. However, it has been more difficult to identify whether broadly neutralizing antibodies circulating in plasma possess shared characteristics among individuals. To address this question, we used affinity chromatography and isoelectric focusing to fractionate plasma immunoglobulin from 10 HIV-1-infected subjects (5 subjects with broad HIV-1 neutralizing activity and 5 controls). We find that plasma neutralizing activity typically partitions into at least two subsets of antibodies. Antibodies with restricted neutralization breadth have relatively neutral isoelectric points and preferentially bind to envelope monomers and trimers versus core antigens from which variable loops and other domains have been deleted. In comparison, broadly neutralizing antibodies account for a minor fraction of the total anti-envelope response. They are consistently distinguished by more basic isoelectric points and specificity for epitopes shared by monomeric gp120, gp120 core, or CD4-induced structures. Such biochemical properties might be exploited to reliably predict or produce broad anti-HIV immunity.

Fig 1

Preparative IEF fractionation of plasma IgG with broad HIV neutralizing activity. Representative data from subject 6 (see the text) are shown. Fractionation of 20 mg IgG was achieved by preparative IEF with a pH range of 6.5 to 9.5 (indicated at bottom), as described in Materials and Methods. An aliquot of each fraction (10 μl) was electrophoresed over an IEF gel with a pH range of 6 to 11 (indicated at left). Immunoglobulin bands were visualized with silver stain. Lanes 1 to 20 correspond to fractions 1 to 20, respectively.

Fig 2

ELISA reactivity patterns of fractionated IgG from subjects with broad or nonbroad plasma HIV neutralizing activity. Whole IgG from each subject was fractionated by preparative IEF (see Materials and Methods). Aliquots (0.5 μg each) of IgG from each fraction were tested by ELISA for reactivity against the indicated HIV antigens. The x axis represents the IEF fractions (spanning a pH gradient of 6.5 to 9.5 from left to right). The left y axis represents ELISA signals expressed as background-corrected optical density at 450 nm (OD₄₅₀) readings/μg IgG. Assays were repeated at least twice. Error bars indicate the high and low values observed. The gp120 core/gp120 core +V3 ratio (right y axis) is calculated from mean absorbance values for each antigen.

Fig 3

IgG Subclass distribution among IEF-fractioned IgG from subjects with broad or nonbroad plasma HIV neutralizing activity. IEF fraction numbers are indicated on the x axis. All fractions were assayed in duplicate by ELISA for IgG1, IgG2, and IgG4 concentrations as described in Materials and Methods. Total IgG was determined by the sum of all IgG subclasses tested. The quantities of IgG subclasses in each fraction were extrapolated from the ELISAs and used to calculate percentages of total IgG (y axis).

Fig 4

Neutralization patterns of IEF-fractioned IgG from subjects with broad or nonbroad plasma HIV neutralizing activity. IEF fractions are indicated on the x axis; the pH gradient of 6.5 to 9.5 spans the fractions from left to right. IgG from each fraction was tested for neutralizing activity against the indicated pseudoviruses as described in Materials and Methods. SF162.LS is a tier 1 pseudovirus; the rest fall within the tier 2 category. Assays were run in duplicate. Mean infectivity values were used to calculate IC₅₀ titers (left y axis). The maximum IgG concentrations tested per fraction are shown on the right y axis. The absence of a symbol for any pseudovirus-fraction pair indicates that an IC₅₀ was not achieved at the highest testable IgG concentration in the fraction.

Fig 5

Neutralization patterns of gp120-affinity-purified, IEF-fractioned IgG from subjects with broad HIV neutralizing activity. IEF fractions are shown on the x axis. A pH gradient of 6.5 to 9.5 spans the fractions from left to right. IgG from each fraction was tested for neutralizing activity against the indicated pseudoviruses as described in Materials and Methods. Assays were run in duplicate. Mean infectivity values were used to calculate IC₅₀ titers (left y axis). The maximum IgG concentrations tested per fraction are shown on the right y axis. The absence of a symbol for any pseudovirus-fraction pair indicates that an IC₅₀ was not achieved at the highest testable IgG concentration in that fraction.

Fig 6

Neutralization activity of basic versus neutral anti-gp120 IgG. Data from Fig. 5 were reanalyzed to calculate the mean IC₅₀ values for the tier 2 virus SC422661.8, RHPA4259.7, or REJO4541.76 among pools of the basic fractions (13 to 17) versus the more neutral fractions (3 to 7). Mean IC₅₀ values for the two pools were compared by paired t test (shown). A P value < 0.05 was taken as significant. Subject 6 was not evaluated due to restricted neutralization breadth among IEF fractions.

Fig 7

Neutralization patterns of HIV envelope-affinity purified, IEF-fractioned IgG from subjects with broad HIV neutralizing activity. The affinity handles used to capture IgG (see Materials and Methods) are shown at top. IEF fractions are shown on the x axis. A pH gradient of 6.5 to 9.5 spans the fractions from left to right. IgG from each fraction was tested for neutralizing activity against the indicated pseudoviruses as described in Materials and Methods. Assays were run in duplicate (except for gp120 core-purified material from subject 1). Mean infectivity values were used to calculate IC₅₀ titers (left y axis). The maximum IgG concentrations tested per fraction are shown on the right y axis. Fractions derived from subject 1 were tested as singletons due to the paucity of sample.