A mammalian cell based FACS-panning platform for the selection of HIV-1 envelopes for vaccine development

An increasing number of broadly neutralizing monoclonal antibodies (bnMAb) against the HIV-1 envelope (Env) protein has been discovered recently. Despite this progress, vaccination efforts with the aim to re-elicit bnMAbs that provide protective immunity have failed so far. Herein, we describe the development of a mammalian cell based FACS-panning method in which bnMAbs are used as tools to select surface-exposed envelope variants according to their binding affinity. For that purpose, an HIV-1 derived lentiviral vector was developed to infect HEK293T cells at low multiplicity of infection (MOI) in order to link Env phenotype and genotype. For proof of principle, a gp145 Env model-library was established in which the complete V3 domain was substituted by five strain specific V3 loop sequences with known binding affinities to nMAb 447-52D, respectively. Env genes were recovered from selected cells by PCR, subcloned into a lentiviral vector (i) to determine and quantify the enrichment nMAb binders and (ii) to generate a new batch of transduction competent particles. After 2 selection cycles the Env variant with highest affinity was enriched 20-fold and represented 80% of the remaining Env population. Exploiting the recently described bnMAbs, this procedure might prove useful in selecting Env proteins from large Env libraries with the potential to elicit bnMAbs when used as vaccine candidates

Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88

The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes and organic acids, particularly citric acid. We sequenced the 33.9-megabase genome of A. niger CBS 513.88, the ancestor of currently used enzyme production strains. A high level of synteny was observed with other aspergilli sequenced. Strong function predictions were made for 6,506 of the 14,165 open reading frames identified. A detailed description of the components of the protein secretion pathway was made and striking differences in the hydrolytic enzyme spectra of aspergilli were observed. A reconstructed metabolic network comprising 1,069 unique reactions illustrates the versatile metabolism of A. niger. Noteworthy is the large number of major facilitator superfamily transporters and fungal zinc binuclear cluster transcription factors, and the presence of putative gene clusters for fumonisin and ochratoxin A synthesis

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

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

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

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

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