Isolation of Monoclonal Antibodies with Predetermined Conformational Epitope Specificity

Existing technologies allow isolating antigen-specific monoclonal antibodies (mAbs) from B cells. We devised a direct approach to isolate mAbs with predetermined conformational epitope specificity, using epitope mimetics (mimotopes) that reflect the three-dimensional structure of given antigen subdomains. We performed differential biopanning using bacteriophages encoding random peptide libraries and polyclonal antibodies (Abs) that had been affinity-purified with either native or denatured antigen. This strategy yielded conformational mimotopes. We then generated mimotope-fluorescent protein fusions, which were used as baits to isolate single memory B cells from rhesus monkeys (RMs). To amplify RM immunoglobulin variable regions, we developed RM-specific PCR primers and generated chimeric simian-human mAbs with predicted epitope specificity. We established proof-of-concept of our strategy by isolating mAbs targeting the conformational V3 loop crown of HIV Env; the new mAbs cross-neutralized viruses of different clades. The novel technology allows isolating mAbs from RMs or other hosts given experimental immunogens or infectious agents

Generation of Recombinant Vaccinia Viruses via Green Fluorescent Protein Selection

We developed a rapid method to generate recombinant vaccinia viruses (rVVs) based upon a bicistronic cassette encoding the gene for green fluorescent protein (GFP) and a foreign gene of interest separated by an internal ribosome entry site (IRES). As proof-of-concept, we inserted a mutant env gene of human immunodeficiency virus (HIV) into the cassette, which was cloned into the vaccinia virus (VV) insertion vector pSC59 under the control of the early-late VV synthetic promoter and flanked by disrupted tk gene sequences. To generate rVVs, 293T cells were inoculated with wild-type (wt) VV, followed by transfection of the modified pSC59 vector containing the bicistronic cassette, which allows expression of GFP and the protein of interest. Next, GFP-positive cells were isolated by flow cytometry or by picking under a fluorescent microscope. Thymidine kinase–deficient (Tk−) 143B cells were then exposed to lysates of GFP-positive 293T cells and cultured in the presence of bromodeoxyuridine. This selection allows only Tk− rVV to remain viable. We demonstrated the success of this GFP selection strategy by expressing high levels of mutant HIV Env. Our approach shortens the time needed to generate rVVs and represents a practical approach to generate recombinant proteins

Differential biopanning strategy to select conformational mimotopes of desired specificity.

<p>HIV gp160 was bound to ELISA plates under native or denaturing conditions. After incubation with serum of monkey RKl-8 (which was chronically infected with a clade C simian-human immunodeficiency virus (SHIV) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038943#pone.0038943-Humbert1" target="_blank">[25]</a> and had high titers of anti-HIV nAbs) followed by washing, Abs were eluted. Abs eluted from the native protein antigen (“conformational” Abs) were used for the positive selection rounds; Abs eluted from the denatured protein (“linear” Abs) were used for the negative rounds. A total of three biopanning rounds were performed and mimotopes selected by this strategy were tested for specificity and conformation dependence with rhesus monkey sera.</p

Strategy of isolation of epitope-specific Abs.

<p>After differential biopanning of phage display peptide library, mimotopes representing conformational epitopes are sequenced, and the inserts plus M13 flanking sequences are cloned into a bacterial expression vector encoding fluorescent protein. After purification of the resulting mimotope fusion proteins, binding assays to assure maintenance of the correct three-dimensional structure of mimotope insert are conducted. This is followed by single-cell sorting for specific IgG-positive memory B cells, single-cell RT-PCR and cloning of RM V_L and V_H regions into pFUSE2-type vectors encoding the backbone of human IgG1.</p

Isolation of single V3 loop-specific memory B cells by FACSorting.

<p>Mimotope-specific memory B cells were selected by the following phenotype: CD3⁻/CD19⁺/CD27⁺/IgG⁺/mWasabi-mimotope⁺ and were sorted into individual wells of a 96-well PCR plate. The consecutive number of gates is shown in the left upper corner of each gate.</p

Neutralization activity of mAbs 33B2 and 33C6.

<p>Neutralization assays were performed in M7-Luc cells (for SHIVs) or TZM-bl cells (for HIVs) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038943#pone.0038943-Montefiori1" target="_blank">[33]</a> unless otherwise mentioned; values in brackets show neutralization assays with A3R5.7 cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038943#pone.0038943-Edmonds1" target="_blank">[36]</a>. *Indicates HIV neutralization in M7-Luc assay. SHIV, simian-human immunodeficiency virus. ND, not determined.</p

Properties of mAbs 33B2 and 33C6.

<p>(A) Binding to envelope proteins of different HIV clades. Plates were coated with Env proteins and probed with newly isolated mAbs along with isotype controls (positive control, HGN194 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038943#pone.0038943-Corti2" target="_blank">[16]</a>; negative control, Fm-6 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038943#pone.0038943-Sui1" target="_blank">[37]</a>) and cognate rhesus monkey (RJa-9) serum. HIV Env proteins were derived from the following strains: clade A, UG37; B, BaL and IIIB; C, CN54 and 1157ip (the latter is a simian-human immunodeficiency virus (SHIV) strain encoding an HIV clade C envelope <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038943#pone.0038943-Humbert1" target="_blank">[25]</a>; D, UG21. SIVmne gp160 was used as negative control. (B) Epitope mapping. Bars show Ab binding by ELISA to consensus HIV clade C peptides representing the V3 loop region. The control peptide represents the scrambled C-terminal sequence of SHIV-1157ip gp120. (C) Virion binding assay. MAbs 33B2 and 33C6 along with negative control (Fm-6) and positive control (HGN194) were captured on the plate coated with goat anti-human IgG Fc-specific Ab and then exposed to SHIV-1157ipEL-p <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038943#pone.0038943-Siddappa1" target="_blank">[19]</a> virions. The amount of virus bound to mAbs was assessed by p27 Gag ELISA. (D) Binding of mAbs 33B2 and 33C6 with native and denatured HIV_CN54 gp120. Plates were coated with native, reduced or reduced/denatured HIV Env protein and probed with 33B2 and 33C6 along with Fm-6 (negative control mAb) as well as serum of HIV-positive individual (positive control), and serum of HIV-negative individual (negative control). We used <i>tris</i>(2-carboxyethyl)phosphine (TCEP) to reduce gp120 and TCEP + SDS to reduce and denature, respectively (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038943#s4" target="_blank">Methods</a>). Each data point represents the mean ± s.e.m. (n  = 3). Experiments were repeated 3 times.</p

Properties of V3 loop mimotopes.

<p>(A) Binding of rhesus monkey RKl-8 serum Abs to native and denatured mimotopes. Wild-type phages (WT) were used as negative control, an anti-phage Ab (anti-M13 pIII) as positive control to show correct phage immobilization. Each data point represents the mean ± s.e.m. (n  = 3). *Mimotope selected for B-cell sorting. (B) Sequence alignment of V3 loop region of gp160 and mimotopes isolated by differential biopanning. Amino acid colors indicate: blue, basic; green, non-polar (hydrophobic); orange, polar (uncharged); magenta (acidic). (C) Binding of RM sera to different V3 mimotopes. Plates were coated with mWasabi-mimotope fusion proteins and parental mWasabi as control and then probed with sera of rhesus monkeys with high nAb titers. Serum from monkey RKl-8 served as positive control; serum from a naïve animal was used as negative control in all experiments. Each data point represents the mean ± s.e.m. (n  = 3). (D) Structural superimposition of Tc.2 mimotope and V3 peptide obtained by <i>in silico</i> molecular modeling. Tc.2 mimotope was modeled using the published V3 peptide structure in complex with Fab2557 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038943#pone.0038943-Jiang1" target="_blank">[13]</a>. The original V3 loop is shown in blue, the Tc.2 mimotope in orange. The cysteine residues and disulfide bridge are in green.</p

VH and VL CDR sequences of V3-specific mAbs.

<p>(A) 33B2 and 33C6 VH CDR amino acid sequences. (B) 33B2 and 33C6 VL CDR amino acid sequences.</p

Genetic characteristics of V3-specific mAbs.

<p>Genetic characteristics of V3-specific mAbs.</p