Affinity analysis of 447-52D antibody to individual members of the Env/V3 library.

<p>HEK293T cells were separately transduced at low MOI (0.1) by one of the pQL9 derived lentiviral particles expressing the indicated chimeric Env/V3-variant, respectively. 48 h post transduction, cells were stained with MAb 447-52D and an APC-labeled secondary antibody. The mean values of two independent experiments corrected for equal GFP expression are shown. <b>A</b> A time-course with 447-52D antibody (10 µg/mL) was performed to record the incubation-time needed for equilibrium binding. FACS data are expressed as the MFI for every member of the model library at the different incubation times, respectively. <b>B</b> 447-52D antibody concentrations were serially diluted and incubated on infected cells at equilibrium incubation-time (1 h at 4°C) to obtain a concentration dependent binding profile. <b>C</b> Cell populations stained with 0.1 µg/mL 447-52D antibodies are depicted separately in order to highlight the differential binding at this concentration. <b>D</b> Dissociation constants (K_D) were calculated from the antibody-titration curves shown in (B) based on the µM concentrations derived from the 447-52D molecular weight and therefore expressed as Kd (µM). Data points were fitted by nonlinear least squares regression (One site; Fit total and nonspecific binding, Graphpad Prism 5), and the resulting K_d as well as R² values are listed (#, non-calculable).</p

Enrichment of Env/V3-variant MN.^a

a<p>Values obtained by qPCR.</p>b<p>Calculations were made according to the formula <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109196#pone.0109196-Khare1" target="_blank">[72]</a>.</p><p># cycle of panning.</p><p>Enrichment of Env/V3-variant MN.^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109196#nt101" target="_blank">a</a>}</p

FACS-panning: gating strategy.

<p>HEK293T (13×10⁶) cells were transduced at low MOI (0.1) (<b>A</b>) by lentivirus particles derived from a plasmid mixture containing equal amounts of all Env/V3 variants and (<b>B</b>) by separately lentiviral particles each encoding only one of the chimeric Env/V3-variants, respectively. Equal amounts of transduced cells were harvested 72 h post transduction and were analysed by FACS. <b>A</b> One typical sorting experiment is shown to illustrate the FACS gating and sorting strategy: 50,000 events were recorded and a series of hierarchical gates were applied to isolate single cells. GFP positive cells were gated within their main population (Gate 5) to exclude multiply infected cells (high GFP) and background noise (low GFP). The GFP main population was subsequently gated for strong 447-52D antibody binding (high APC) in relation to Env expression (coupled to the GFP signal). Therefore, a triangle shaped Gate (Gate 6) was chosen. The complete Gate hierarchy starting with all events is illustrated. <b>B</b> Evaluation of the triangle-gate strategy: HEK293T cells (13×10⁶) were transduced at low MOI (0.1) with each pQL9 Env/V3-virus variant, respectively. Cells were stained with 447-52D antibody and analyzed by FACS according to the gating strategy described in A. Cells that appeared in Gate 5 were further analyzed by calculating a linear regression curve (purple). Slope values calculated for representative variants Env/V3-MN, Env/V3-HXB2, and Env/V3-SF33 are indicated. The triangle shaped Gate 6 is shown in a log-scale dot plot. The numbers of events counted for Gate 5 and Gate 6 are shown below each plot, respectively. Decreasing amounts of cells equal decreasing affinities of Env, as detected in the sorting Gate 6, ranging from 10 counts of Env/V3-MN, 2 of Env/V3-HXB2 and 0 of Env/V3-SF33, respectively.</p

FACS-panning with improved pQL11 based Env/V3 model-library.

<p><b>A</b> Schematic overview of the new pQL11 plasmid <b>B</b> HEK293T cells were infected at low MOI (0,1) of “linked” pQL11 based chimeric Env/V3-model-library viruses and were applied to a FACS-panning procedure 48 h after infection (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109196#s4" target="_blank">Material and Methods</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109196#pone-0109196-g001" target="_blank">Figure 1</a>/<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109196#pone-0109196-g005" target="_blank">5</a>). Env genes were amplified from genomic DNA of collected cells, cloned into fresh pQL11 and were analyzed by qPCR. New virus batches were produced with the resulting new pQL11 Env-library mixtures for infections of fresh cells, thus entering another cycle of selection. The mean values of three independent experiments are shown. Statistics were calculated using the 1-way-ANOVA- (testing, whether mean values differ) and “Dunnett's” post-test (testing, which mean values differ: * P<0.05; ** P<0.01; *** P<0.001). The relative amounts per variant of the input mixture, 1^st and 2^nd cycle are shown as analyzed by qPCR (Efficiency∧^-Ct as % of each variant). The total amounts of pQL11 Env/V3-plasmids in every sample was tested by a reference primer pair that amplifies a sequence independent of the Env genes, respectively (data not shown).</p

Schematic overview of the FACS-panning procedure.

<p>HEK293T cells are transfected with a plasmid mix comprising (i) a lentiviral vector plasmid (pQL9/11) encoding the Env/V3 model library, (ii) a packaging construct (pTNpack) as well as (iii) pVSV-G to render released particles transduction competent (1). Linkage between geno- and phenotype from the initially “unlinked” virus library is achieved by low MOI infection of new cells (2). Antibodies (e.g. bnMAb) are applied to bind cells expressing the various envelopes, respectively (3). Envelope expressing cells displaying the highest MFI to the bnMAb and an internal control (GFP) are selected by a FACS-sorting procedure (4). Cells are collected and lysed prior to amplifying the envelope genes from genomic DNA (5). The recovered envelope genes are cloned into pQL9/11 and analyzed by sequencing and realtime-PCR (6). Fresh cells are transfected (together with pTN pack and pVSV-G) to produce new virus for Low MOI infection of fresh cells, thus entering a new cycle of selection.</p

Expression levels of chimeric Env/V3 and GFP.

<p>HEK293T (3×10⁵) cells were separately transduced at MOI 1 by one of the pQL9 derived transduction competent particles encoding one of the chimeric Env/V3-variants, respectively. 72 h after transduction, cells were harvested and stained with an APC-labeled 5F3 antibody binding a constant region within the extracellular gp41 mojety and compared to GFP mediated fluorescence (Pearson correlation p<0,01 for each variant). <b>A</b> FACS analyses are depicted as the mean fluorescence intensity (MFI) of APC labeled 5F3 antibody- and GFP-signals for all chimeric Env/V3 variants, respectively. <b>B</b> The MFI ratios of 5F3 to GFP signals were calculated, respectively.</p

Schematic overview of the QL cloning procedure.

<p>An envelope gene or an envelope library is amplified with primers to introduce flanking Esp3I restriction sites enabling the generation of a 5′ NcoI and a 3′ Xho sitey (A; top). The envelope gene or an envelope library is incubated together with pQL9/11 in a one-tube reaction with Esp3I and T4-Ligase. Compatible “sticky-ends” (equally colored) can be ligated successfully, direct proper orientation and mediating resistance for further cleavage (A). Following transformation of CcdB sensitive bacteria, only recipients bearing a plasmid without CcdB are able to form colonies in the presence of ampicillin. (B) The lentiviral vector construct pQL9 comprises (i) 5′LTR (Long terminal repeat), (ii) EF1α (human promotor), (iii) GFP (marker gene), (iv) an IRES (internal ribosome entry site), (v) a CcdB positive selection marker <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109196#pone.0109196-Li1" target="_blank">[58]</a>, and (vi) a 3′LTR sequence.</p

VHH repertoires of naïve and protein-immunized llamas.

<p>VHH sequences from immunized llamas 8 and 9 and seven naïve llamas were amplified from their respective phagemid libraries by PCR using primers specific to the 5′ and 3′ conserved regions of the VHH and subjected to 454 sequencing. (<b>A</b>) Unique sequences generated from the indicated llama phage library were used to build end-joining network diagrams with significantly more linkages (P = 0.001) in the naive llamas versus (<b>B</b>) immunized llamas. (<b>C</b>) Shared percentage identities with neutralizing VHH J3 and divergence from its inferred V gene Vt were calculated for all unique sequences from the control naïve llamas, and the J3-source llama 8. The left hand panel shows percentage identity for all sequences from naïve 3 plotted against divergence from Vt. The right panel shows percentage identity for all sequences from llama 8 plotted against divergence from Vt. The horizontal dotted line on each panel indicates the percentage identity shared by Vt and J3 and the vertical dotted line indicates the divergence of J3 from Vt, (<b>D</b>) Shared percentage identities with neutralizing VHH 3E3 and divergence from its inferred V gene Ve were calculated for all unique sequences from the control naïve llamas, and the 3E3-source llama 9. The left hand panel shows percentage identity for all sequences from naïve 3 plotted against divergence from Ve. The right panel shows percentage identity for all sequences from llama 9 plotted against divergence from Ve. The horizontal dotted line on each panel indicates the percentage identity shared by Ve and 3E3 and the vertical dotted line indicated the divergence of 3E3 from Ve.</p

Anti-HIV activity of germ line V gene VHH.

<p>GL VHH comprising inferred germ line V gene paired with the mature VHH CDR3 and J region were produced. GL VHH binding to (<b>A</b>) clade B immunogen R2 gp140 and (<b>B</b>) clade C immunogen 96ZM9651.02 gp140 was assessed by ELISA as per the materials and methods. GL VHH neutralization of (<b>C</b>) R2 pseudovirus and (<b>D</b>) 96ZM651.02 pseudovirus was assessed in TZMbl assay as per the materials and methods. (<b>E</b>) Unique sequences from llamas 1 and 3 at both timepoints were filtered for B9, A14 and B21 germ line V gene usage (Vg and Vu) respectively. The y-axis shows the percentage of sequences within each subset with identical residues to each VHH at each individual CDR3 position (and the preceeding three V gene residues −1,−2,−3 = CAT/CNA) indicated on the x-axis. (<b>F, G, H</b>) The number of unique sequences within the subsets which share exact runs of CDR3 residues are plotted on the y-axis against runs of increasing CDR3 length on the x-axis for (<b>F</b>) B9, (<b>G</b>) A14 and (<b>H</b>) B21 at the time points indicated in the legend.</p

Combined breadth and potency of anti-CD4bs VHH.

<p>(A) IC50 values in µg/ml against the HIV strains indicated in the left-hand column on TZM-bl cells. VHH were titrated five-fold in duplicate both individually (from 10 µg/ml) and in combination with one another in the same plate for each virus. IC50 values of less than 0.1 are color-coded in dark red, those between 0.1 and 1 in red, those between 1 and 10 in orange and those above 10 in yellow. (B) IC50 values in µg/ml against each of the HIV strains indicated on the X-axis are shown for J3 or the combined VHH according to the color-coded legend. (C) Each spoke represents a strain of HIV neutralized by J3 or the combined VHH. Strains from different clades and CRF are color-coded according to the labels. The outer circle represents an IC50 of <1 µg/ml, the inner circle <5 µg/ml and the centre of the circle 50 µg/ml.</p