Polyreactivity increases the apparent affinity of anti-HIV antibodies by heteroligation

Abstract

During immune responses, antibodies are selected for their ability to bind to foreign antigens with high affinity, in part by their ability to undergo homotypic bivalent binding. However, this type of binding is not always possible. For example, the small number of gp140 glycoprotein spikes displayed on the surface of the human immunodeficiency virus (HIV) disfavours homotypic bivalent antibody binding. Here we show that during the human antibody response to HIV, somatic mutations that increase antibody affinity also increase breadth and neutralizing potency. Surprisingly, the responding naive and memory B cells produce polyreactive antibodies, which are capable of bivalent heteroligation between one high-affinity anti-HIV-gp140 combining site and a second low-affinity site on another molecular structure on HIV. Although cross-reactivity to self-antigens or polyreactivity is strongly selected against during B-cell development, it is a common serologic feature of certain infections in humans, including HIV, Epstein-Barr virus and hepatitis C virus. Seventy-five per cent of the 134 monoclonal anti-HIV-gp140 antibodies cloned from six patients with high titres of neutralizing antibodies are polyreactive. Despite the low affinity of the polyreactive combining site, heteroligation demonstrably increases the apparent affinity of polyreactive antibodies to HIV.

Figure 1. Polyreactivity, anti-cardiolipin and HEp-2 ELISAs

a, ELISAs measuring the reactivity of 4E10 (ref. 23), b12 (ref. 24), 2F5 (ref. 25) and 2G12 (ref. 26) against double-stranded DNA, single-stranded DNA, insulin and lipopolysaccharide. Dotted lines represent the positive control antibody ED38 (ref. 27). Horizontal lines show cut-off OD_{405 nm} for positive reactivity. Green and red lines show the negative and low positive control antibodies, respectively^,. b, As in a but for IgG antibodies cloned from gp140⁺ cells from patients 1–4 and for gp140⁻ cells from patients 2 and 3. Pie charts summarize the frequency of polyreactive (black) and non-polyreactive (white) IgG⁺ clones, and for all pooled gp140⁺, gp140⁻ and previously published control IgG antibodies (HC-IgGm) (right). P values are in comparison with pooled gp140⁻ IgGs or between gp140⁺ and gp140⁻ for patients 2 and 3 as indicated. c, Graphs show anti-cardiolipin ELISAs for the same antibodies as in b. Orange and blue lines show the positive control antibodies 4E10 and 2F5 (refs 23, 25), respectively. Pie charts summarize the frequency of cardiolipin reactive (black) and non-reactive (white) IgGs. P values are in comparison with pooled gp140⁻ IgGs (right) or between gp140⁺ and gp140⁻ for patients 2 and 3 as indicated. d, Same as b except for HEp-2 ELISAs.

Figure 2. IgH chain gene features and reactivity

a, Pie charts summarize polyreactivity, anti-cardiolipin and HEp-2 reactivity of gp140⁺ antibodies grouped by antibody specificity for gp41, gp120, gp120^core, CD4bs, variable loops and CD4i. Reactive (black) and non-reactive (white) IgGs are shown; numbers in the centre indicate number of antibodies tested. P values are in comparison with gp140⁻ IgGs unless otherwise indicated. b, Polyreactivity of all gp140-, gp120- and gp41-specific IgG antibodies grouped according to IgH CDR3 length, IgH CDR3-positive charges, IgH CDR3 hydropathy (GRAVY) or number of VH mutations.

Figure 3. Reactivity of reverted antibodies

a, ELISAs against gp140 for 20 selected polyreactive and non-polyreactive antibodies (Supplementary Table 2), before (mutated) and after (reverted) reversion. The red line indicates the reactivity of the control b12 antibody. b, Anti-double-stranded DNA ELISAs (as example for polyreactivity) for the mutated and reverted antibodies that were initially polyreactive (top) or non-polyreactive (bottom). Numbers on the right indicate antibodies in reactive or non-reactive categories before and after reversion. c, As in b except for HEp-2 reactivity. Positive and negative control antibodies are as in Fig. 1.

Figure 4. Heteroligation

a, Representation of bivalent and monovalent homotypic binding versus heteroligation on high- and low-density gp120, high-density KLH and mixed chips. b, Apparent K_a (K_a^app) as measured by SPR on chips derivatized with high or low concentrations of gp120 or high concentrations of KLH, or a combination of low-gp120 and high-KLH. Numbers indicate the fold enhancement that can be attributed to heteroligation. See also Supplementary Table 4. Error bars, s.e.m. c, ELISA binding studies for IgG and corresponding Fabs on HIV-BaL. d, Solution competition-binding assay for fluorescent BaL virus using gp120 or KLH as competitors. Coloured lines show fitted curves. The y axis indicates the relative mean fluorescence index (rMFI), the x axis the competitor concentration in micromoles. Gp120 and KLH concentrations required to achieve 50% binding inhibition (IC₅₀) are indicated. e, SPR sensorgrams for binding to BaL virus or gp120 protein to IgG antibody-immobilized sensor chips as illustrated by the schematic. RU, response units. f, Apparent K_a (K_a^app) for gp120 protein as measured by SPR. Error bars, s.e.m. g, As f but for the relative K_a to BaL virus (K_a^rel_BaL).