Fig 1.
Diagram of the affinity maturation process.
1) Error-prone PCR introduces mutations in the original single chain antibody (scFv) gene. 2) A library of ~ 1x107 diversity is created by co-transforming the error-prone PCR amplicon and the linearized display vector in yeast. 3) The portion of the library with the highest level of yeast display (phycoerythrin -PE- fluorescence) and antigen binding (Alexa 633 fluorescence) is sorted by flow cytometry (2–5% of the entire population). Sorting is repeated 3 to 4 times at progressively decreasing antigen concentrations. 4) Single clones in the last sorted population are analyzed one by one using sub-saturating amounts of antigen. 5) The best binder (orange star) is identified by sequencing. Steps 1 through 5 are repeated until sorting at 1 nM antigen concentration is possible.
Fig 2.
Comparison of original and affinity matured yeast-displayed scFv antibodies.
(A) The yeast-associated fluorescence after incubation with biotinylated F1V and staining with streptavidin-APC (allophycocyanin) was measured by flow cytometry. Mean fluorescence intensity values at antigen concentrations of 500, 50, and 5 nM, and corresponding standard deviations (error bars) are reported for the original (αF1sc 2 and 8) and the affinity matured (αF1sc AM2 and AM8) antibody clones. 500 nM biotinylated myoglobin was used as a negative control antigen. (B and C) The same data reported in A were fit in the one-site binding equation [AB = ABmax*[Ag]/(KD+[Ag])] and KD resulting from the fit are indicated. The nomenclature EPX-Y in the legend denotes the error-prone library number (X) and the best clone number (Y) identified from this library. The best affinity matured αF1sc 2 clone (indicated for simplicity as αF1sc AM2 in A) was clone 18 from error-prone library 3 (αF1sc 2 EP3-18). The best affinity matured αF1sc 8 clone (indicated as αF1sc AM8 in A) was clone 24 from error-prone library 2 (αF1sc 8 EP2-24).
Fig 3.
Antibody stability by dynamic light scattering analysis.
The hydrodynamic radius of anti-F1 affinity matured antibody 2 and 8 (αF1Ig AM2, orange; αF1Ig AM8, green) was ~14 nm and did not change after 150 days storage at 37°C in PBS. These results indicate lack of protein aggregation in the conditions tested. A mouse anti-lysozyme IgG stored at 4°C for the duration of the experiment was used as a control (blue). In this figure, two per moving average trendline is indicated as dotted lines.
Table 1.
Affinities of anti-F1 antibodies for F1 antigen.
Fig 4.
Kinetic study of purified anti-F1 IgGs by ELISA and SPR.
(A) ELISA data obtained for original anti-F1 IgGs (αF1Ig 2 and 8) or affinity matured (αF1Ig AM2 and 8) antibodies are shown. Experiments were performed in triplicate and data points averaged. The standard deviation of each data point was calculated and shown as the error bar. Data were fitted to one-site specific binding equation. AB = antibody-F1 binding; ABmax = antibody-F1 binding at saturation; KD = dissociation constant = half saturating antibody concentration ([Ab]). (B) Representative surface plasmon resonance sensograms for each parental and affinity matured antibody are shown.
Fig 5.
The anti-F1 monoclonal antibodies are opsonic in nature.
As demonstrated in an in vitro gentamicin protection assay the mouse derived α-F1 mAb (F1-04-A-G1) and the human derived αF1sc AM2 and αF1sc AM8 mAbs were opsonic. RAW264.7 cells were infected with Y. pestis CO92 pgm- pPst- at an MOI of approximately 10 CFU. (*) indicates P <0.0001 when compared to Y. pestis (no antibody) alone and P < 0.0001 when compared to anti-influenza M2 (αM2 IgG Z3) negative control, (**) indicates P = 0.0007 when compared to Y. pestis alone and P < 0.0001 when compared to anti-influenza M2 negative control. This is the average of 2 experiments and representative of 5 experiments total.
Table 2.
In vivo experiments using the BALB/c mouse model of pneumonic plague to demonstrate protection of antibody therapy when given as pre-exposure prophylaxis.