Llama antibody fragments with cross-subtype human immunodeficiency virus type 1 (HIV-1)-neutralizing properties and high affinity for HIV-1 gp120

Abstract

Members of the Camelidae family produce immunoglobulins devoid of light chains. We have characterized variable domains of these heavy chain antibodies, the VHH, from llamas immunized with human immunodeficiency virus type 1 (HIV-1) envelope protein gp120 in order to identify VHH that can inhibit HIV-1 infection. To increase the chances of isolating neutralizing VHH, we employed a functional selection approach, involving panning of phage libraries expressing the VHH repertoire on recombinant gp120, followed by a competitive elution with soluble CD4. By immunizing with gp120 derived from an HIV-1 subtype B'/C primary isolate, followed by panning on gp120 from HIV-1 isolates of subtypes A, B, and C, we could select for VHH with cross-subtype neutralizing activity. Three VHH able to neutralize HIV-1 primary isolates of subtypes B and C were characterized. These bound to recombinant gp120 with affinities close to the suggested affinity ceiling for in vivo-maturated antibodies and competed with soluble CD4 for this binding, indicating that their mechanism of neutralization involves interacting with the functional envelope spike prior to binding to CD4. The most potent VHH in terms of low 50% inhibitory concentration (IC(50)) and IC(90) values and cross-subtype reactivity was A12. These results indicate that camelid VHH can be potent HIV-1 entry inhibitors. Since VHH are stable and can be produced at a relatively low cost, they may be considered for applications such as HIV-1 microbicide development. Antienvelope VHH might also prove useful in defining neutralizing and nonneutralizing epitopes on HIV-1 envelope proteins, with implications for HIV-1 vaccine design.

FIG. 1.

(A) Schematic overview of the strategy for isolation of llama VHH targeting the CD4bs of gp120. (B) Titration of eluted phage onto E. coli TG1 cells. Phage bound to gp120 was eluted using sCD4. As a control, elution with BSA was performed in parallel, as was a general elution by low-pH shock using glycine. Shown is a representative titration of eluted phage, where more clones were eluted by sCD4 than by BSA and from which individual VHH were isolated and expressed.

FIG. 2.

VHH and MAb b12 IC₅₀ titers against HIV-1 in TZM-bl cells. VHH and MAb b12 neutralization activity was assessed in the indicated viruses, as described in the text. Rabies virus CVS-11 is pseudotyped with rabies virus G-protein from strain CVS-11 (87). Abbreviations for virus types: TCLA, T-cell-line-adapted isolate; PBMC, PBMC-propagated primary isolate; MC, molecular clone; PV, envelope pseudotyped virus. The column labeled Tier indicates whether the virus is classified as suitable for tier 1, 2, or 3 assessment of neutralizing antibodies (46). •, IC₅₀ >50 μg/ml; nd, not determined; n/a, not applicable. To aid comprehension, the titers have been shaded, with darker colors indicating more potent neutralization.

FIG. 3.

Percentage of HIV-1 isolates neutralized by the VHH and by MAb b12, according to HIV-1 subtype. Virus neutralization was assayed in TZM-bl cells as described in the text. Shown is the percentage of viruses neutralized with an IC₅₀ and IC₉₀ of ≤50 μg/ml.

FIG. 4.

(A) VHH binding to recombinant envelope proteins derived from HIV-1 92UG037 (subtype A), IIIB (subtype B), and 92BR025 (subtype C) in the ELISA. Recombinant envelope proteins were captured by immobilized antibody D7324. Serial dilutions of VHH A12, D7, C8, and a negative control VHH were then added, and binding was detected as described in the text. (B) Dose-dependent competition of VHH A12, D7, and C8 with sCD4 for binding to recombinant envelope proteins in the ELISA. Threefold serial dilutions of VHH were preincubated with IIIB gp120 or 92UG037 gp140 and subsequently incubated with sCD4 precoated on microtiter plates. Envelope protein binding to sCD4 was detected as described in the text. Chemiluminescence was measured, and background subtracted luminescence readings (in relative light units [RLU]) were plotted against VHH concentration. Data points represent the means and bars show the standard deviations of duplicate reactions.

FIG. 5.

VHH and anti-gp120 MAb cross-competition analysis. (A) Dose-dependent competition of VHH A12, D7, and C8 with anti-CD4bs MAbs b12, 654-D, and GP68 for binding to recombinant IIIB gp120 in the ELISA. Serial dilutions of VHH were preincubated with IIIB gp120. Envelope protein binding to human anti-CD4bs MAbs b12, 654-D, and GP68 was detected as described in the text. (B) VHH competition with anti-gp120 MAbs 2G12 (carbohydrate motif), 17b (CD4i), and 447-52D (V3), as well as MAb b12 (CD4bs), for binding to recombinant gp120 in the ELISA. Wells of microtiter plates were coated with VHH A12, D7, and C8, as indicated above each graph. Serial dilutions of each MAb were preincubated with IIIB gp120 and subsequently incubated with the immobilized VHH. Envelope protein binding to VHH was detected as described in the text. (C) VHH competition with each other and with sCD4 for binding to recombinant gp120 in the ELISA. Wells of microtiter plates were coated with VHH A12, D7, and C8, as indicated above each graph. Serial dilutions of each VHH and sCD4 were preincubated with IIIB gp120 and subsequently incubated with the immobilized VHH. Envelope protein binding to VHH was detected as described in the text. Background-subtracted luminescence readings were plotted against MAb concentration. Data points represent the means and bars show the standard deviations of duplicate reactions.