De Novo Isolation & Affinity Maturation of yeast-displayed Virion-binding human fibronectin domains by flow cytometric screening against Virions

Abstract

Background: The promise of biopharmaceuticals comprising one or more binding domains motivates the development of novel methods for de novo isolation and affinity maturation of virion-binding domains. Identifying avenues for overcoming the challenges associated with using virions as screening reagents is paramount given the difficulties associated with obtaining high-purity virus-associated proteins that retain the conformation exhibited on the virion surface.

Results: Fluorescence activated cell sorting (FACS) of 1.5 × 10⁷ clones taken from a naïve yeast surface-displayed human fibronectin domain (Fn3) against whole virions yielded two unique binders to Zika virions. Construction and FACS of site-directed binding loop mutant libraries based on one of these binders yielded multiple progeny clones with enhanced Zika-binding affinities. These affinity-matured clones bound Zika virions with low double- or single-digit nanomolar affinity in ELISA assays, and expressed well as soluble proteins in E. coli shake flask culture, with post-purification yields exceeding 10 mg/L.

Conclusions: FACS of a yeast-displayed binding domain library is an efficient method for de novo isolation of virion-binding domains. Affinities of isolated virion-binding clones are readily enhanced via FACS screening of mutant progeny libraries. Given that most binding domains are compatible with yeast display, the approach taken in this work may be broadly utilized for generating virion-binding domains against many different viruses for use in passive immunotherapy and the prevention of viral infection.

Keywords: AIDS; Flow cytometry; Human Immunodeficiency Virus; Zika virus; antibody engineering; directed evolution; fibronectin; phage display; protein engineering; yeast display.

Fig. 1

Schematic for sandwich detection of yeast surface-displayed Fn3 binding to Zika virions. Anti-Zika human IgG conjugated with Alexa488 enables detection of Fn3-Zika virion binding interaction. Alexa 405-conjugated anti-myc antibody (IgY from chicken) binds to myc tag on Fn3 C-terminus and allows quantification of Fn3 surface display

Fig. 2

Flow cytometry dot plots for FACS enrichment of Zika virion-binding Fn3s. Y-axes denote binding of surface-displayed Fn3s to Zika virions as quantified via sandwich detection using Alexa488-conjugated anti-Zika IgG. Panels, left-to-right, show representative samples (100,000 yeast cells) of yeast populations screened during FACS rounds one, two and three

Fig. 3

Flow cytometric histogram overlay for yeast-displayed Fn3s binding to Zika virions. X-axis denotes binding of surface-displayed Fn3s to Zika virions as quantified via sandwich detection using Alexa488-conjugated anti-Zika IgG. Ab only denotes yeast samples not incubated with Zika virions prior to labeling with Alexa488-conjugated anti-Zika IgG secondary reagent. Histograms contain data for 30,000 analyzed yeast cells per sample

Fig. 4

Representative flow cytometric dot plots for analysis of yeast-displayed Clone71 site-directed saturation mutant libraries. Dot plot data was used to identify libraries containing candidate improved affinity Fn3 mutants. Pink ellipses in dot plots for position 80 and position 82 saturation mutant libraries encase yeast populations lying above the bulk diagonal. Position 85 mutant library (rightmost panel) provides example of saturation mutant library devoid of candidate higher affinity virion binders. Induced yeast were mixed with uninduced yeast at a ratio of 1:10 to prevent ligand (Zika virion) depletion effects. Dot plots contain data for 250,000 analyzed yeast cells

Fig. 5

Flow cytometric histogram overlays for yeast-displayed Fn3s binding to Zika virions. X-axes denote binding of surface-displayed Fn3s to Zika virions as quantified via sandwich detection using Alexa488-conjugated anti-Zika IgG. C71: Clone 71. Ab Only denotes yeast samples incubated with culture supernatant from naïve Vero cells prior to labeling with Alexa488-conjugated anti-Zika IgG secondary reagent. Left panel: Yeast incubated with Zika virion stock diluted 1:5 in PBS/0.3% BSA. Right panel: Yeast incubated with Zika virion stock diluted 1:20 in PBS/0.3% BSA. Histograms contain data for 30,000 analyzed yeast cells per sample

Fig. 6

Denaturing (a) and native (b) PAGE analyses of Zika-binding and negative-control Fn3s. Calculated molecular weight for Fn3s is ~ 13 kDa. Native PAGE analysis indicates that the Clone71 F80P mutant reduces the formation of dimeric and oligomeric Fn3 complexes

Fig. 7

Immobilized Fn3 ELISA assay. a Assay schematic. b Assay absorbance values. C71: Clone 71. ‘C71 Nil Virus’ denotes negative control in which ELISA plate-immobilized C71 Fn3 was incubated with UF conditioned culture media supernatant from naive Vero cells. Error bars denote standard deviations for triplicate absorbance measurements

Fig. 8

Free-in-solution Fn3 ELISA assay. a Assay schematic. b, c Sandwich detection of virions bound by immobilized IgG. ‘Nil Virus’ denotes incubation with blank assay buffer, rather than virions, prior to incubation with Fn3. ‘Nil mAb’ denotes coating of ELISA plate assay wells with BSA rather than anti-Zika IgG. Error bars denote standard deviations for duplicate absorbance measurements