Abstract

Recently, we described llama antibody fragments (VHH) that can neutralize human immunodeficiency virus, type 1 (HIV-1). These VHH were obtained after selective elution of phages carrying an immune library raised against gp120 of HIV-1 subtype B/C CN54 with soluble CD4. We describe here a new, family-specific approach to obtain the largest possible diversity of related VHH that compete with soluble CD4 for binding to the HIV-1 envelope glycoprotein. The creation of this family-specific library of homologous VHH has enabled us to isolate phages carrying similar nucleotide sequences as the parental VHH. These VHH displayed varying binding affinities and neutralization phenotypes to a panel of different strains and subtypes of HIV-1. Sequence analysis of the homologs showed that the C-terminal three amino acids of the CDR3 loop were crucial in determining the specificity of these VHH for different subtype C HIV-1 strains. There was a positive correlation between affinity of VHH binding to gp120 of HIV-1 IIIB and the breadth of neutralization of diverse HIV-1 envelopes. The family-specific approach has therefore allowed us to better understand the interaction of the CD4-binding site antibodies with virus strain specificity and has potential use for the bioengineering of antibodies and HIV-1 vaccine development.

Highlights

  • A summary of the selection conditions used in the biopanning process is shown

  • 2nd round recognized a similar epitope on gp[120]

  • We have successfully used a novel method to create a family-specific library of HIV-1-neutralizing VHHcontaining variants that are homologous to the parental A12/D7 VHH that neutralize a broad spectrum of HIV-1 isolates (30)

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Summary

EXPERIMENTAL PROCEDURES

Recombinant gp[120] Antigen Preparation—Recombinant gp[120] from HIV-1 IIIB 10 ␮g/ml recombinant sCD4 containing the D1–D3 regions of CD4, obtained from the Centralized Facility for AIDS Reagents (National Institute for Biological Standards and Control, Potters Bar, UK), was coated overnight on 96-well Maxisorp plates. These were tested for binding in ELISA and for its neutralization potency in the TZM-bl assay. HIV-1 Neutralization Assays—The neutralizing ability of the VHH was tested against a panel of HIV-1 viruses using a luciferase-based assay in TZM-bl cells (46 – 48), which was obtained through the AIDS Research and Reference Reagent Program, National Institutes of Health, from J. The calculations were performed using the XLFit[4] software (ID Business Solutions, Guildford, UK), and the assay was performed on at least two separate occasions

RESULTS
Summary of outputs from selections
DISCUSSION
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