3.1. Identification of alpaca VHHs with affinity for BoNT/A and BoNT/B proteases
Two alpacas were immunized with recombinant BoNT/A light chain (A-Lc) protease achieving titers exceeding 106. A VHH-display library was produced from B cells of the immunized alpacas in which the VHH coding DNA was amplified by PCR and ligated into an M13 phage display vector. Several hundred thousand independent clones were obtained, amplified and the phage was panned through multiple cycles for binding to A-Lc. After characterization of more than 100 clones recognizing A-Lc by ELISA, 36 clones with apparently unique BstN1 fingerprints were selected for sequencing. Alignment of these clones together with 17 random alpaca VHHs (not shown) revealed that all A-Lc binding VHHs fell into two clearly distinct homology groups. Four representatives of the first homology group (ALc-A6, D4, E3 and G6, Genbank accession numbers FJ643071-4) were selected for expression based on sequence divergence and tested for A-Lc binding by dilution ELISA. Binding to A-Lc was detected for all four VHHs when diluted to 10 nM or less (not shown). VHH ALc-D4 had the highest apparent affinity and A-Lc could be detected by ELISA when diluted to 0.5 nM. Most of the A-Lc binding VHHs isolated were members of the second VHH homology group. Surprisingly, members of this group always contained a single amber codon within the VHH coding region that was not always at the same amino acid position. Functional VHH display of these VHHs would occur, probably at a lower level, since the phage was produced within an amber suppressor strain of E. coli. A representative of the most frequently obtained VHH sequence in this homology group (ALc-B8, Genbank accession number FJ643070) was selected for further study.
The same two alpacas were immunized about a year later with recombinant BoNT/B Lc (B-Lc) and developed anti-B-Lc titers near 106. Prior to B cell preparation, the animals received a final boost with both recombinant A-Lc and B-Lc. A phage display library was prepared as above and panned for B-Lc binding. Characterization of more than 100 clones found to be positive for B-Lc binding identified 15 unique clones and the VHH coding regions were sequenced. Homology analysis revealed that all clones were contained within two closely related groups. Within each homology group, all unique family members encode VHHs differing by only 1–5 amino acids involving conservative amino acid variations. One representative of each group (BLc-B10 and BLc-C3, Genbank accession numbers GU168771-2) was selected for further study.
In a separate effort, panning cycles were alternately performed with B-Lc and A-Lc to select for clones expressing VHHs that bind to both A-Lc and B-Lc. Following extensive screening, only a single clone was found that expressed phage binding to both A-Lc and B-Lc. Bacteria from this clone were found to harbor two plasmids. One of the plasmids was a typical phagemid encoding a VHH similar to BLc-B10. The second plasmid was also a phagemid but it was unstable in the bacteria and contained a significant deletion that included part of the gene III coding region. This plasmid contained a new VHH coding DNA (ALc-H7, Genbank accession numbers FJ643075) that was specific for A-Lc (see below). Thus, although the panning process selected for a bacterial clone expressing VHH with binding specificity for both A-Lc and B-Lc, the dual specificity was the result of a very unusual clone harboring two phagemids, each encoding a different VHH, each specific for a different Lc serotype. This indicates that single VHHs able to bind to both Lc serotypes were likely not present in our library and are either rare or non-existent within the alpacas.
3.2. Soluble, recombinant VHHs bind A-Lc or B-Lc
DNA encoding the three selected VHHs shown to bind A-Lc (ALc-B8, ALc-D4 and ALc-H7) and the two selected VHHs shown to bind B-Lc (BLc-B10 and BLc-C3) were each cloned into an E. coli expression vector. The amino acid sequences of the VHHs are shown in . An amber codon present in ALc-B8 at position 7 () was changed to a glutamine codon, the amino acid almost always found in VHHs at this position. Each VHH was expressed in E. coli and purified.
Fig. 1 Characterization of the five selected VHH anti-BoNT Lc binding proteins. (A) Amino acid sequences of the five selected anti-BoNT Lc VHH domains aligned for maximum homology. (B) Dilution ELISA to measure binding of five VHHs to plates coated by 5 ug/ml (more ...)
The five selected VHHs were titered by ELISA for their recognition of BoNT A-Lc or B-Lc. As shown in , VHHs ALc-B8, ALc-D4 and ALc-H7 were entirely specific for A-Lc. ALc-B8 and ALc-H7 have the highest apparent affinity for A-Lc and binding to A-Lc can easily be detected when these VHHs have been diluted to sub-nanomolar concentrations. VHH BLc-B10 and BLc-C3 were entirely specific for B-Lc and BLc-B10 had significantly higher apparent affinity than BLc-C3. Based on this ELISA, BLc-B10 displayed a high apparent affinity for B-Lc that was equivalent or better to that of ALc-B8 and ALc-H7 for A-Lc.
3.3. Two anti-BoNT/A-Lc VHHs potently inhibit the protease activity
Each of the five anti-BoNT Lc VHHs, purified from recombinant E. coli, was tested for its effect on the proteolytic function of the protease to which it binds. The two B-Lc binding VHHs had no detectable inhibitory effect on B-Lc proteolytic activity (not shown). VHHs ALc-B8 and ALc-H7 were potent inhibitors of A-Lc protease. As shown in , both VHHs inhibited A-Lc protease in a FRET-based SNAP25 cleavage assay. Both VHHs inhibited 2 nM BoNT/A Lc with IC50 values in the range of 1.6–2.5 nM providing evidence for a near stoichiometric inhibition of the protease. VHH ALc-D4 did not significantly inhibit the A-Lc protease activity in the FRET based assay.
Fig. 2 Two VHHs potently inhibit BoNT/A-Lc protease activity as measured by the FRET assay. Inhibition dose–response curve obtained with Prism 4™ software for VHH ALc-B8 (■) and ALc-H7 (▲) inhibition of BoNT/A-Lc. Values shown (more ...)
The two VHHs showing strong inhibition of A-Lc protease activity, ALc-B8 and ALc-H7, differ at 31 amino acid positions and the CDR3 differs in size by 4 amino acids. Despite this, B8 and H7 cluster together when aligned with random VHH sequences indicating they may be related (not shown). A competition ELISA was performed in which VHHs having a myc tag were tested for binding to A-Lc in the presence of 10× or 100× excess of VHHs having an E-tag, or vice versa. The data showed that a 100-fold excess of ALc-B8 or ALc-H7 reduced binding of the other to A-Lc by >80% (not shown), demonstrating that these VHHs share the same, or closely apposed, epitopes on A-Lc. VHH ALc-D4 did not compete for the binding of ALc-B8 or ALc-H7 to A-Lc, or vice versa. Similar competition experiments with BLc-B10 and BLc-C3 found that these VHHs do not compete with the other for binding to B-Lc.
3.4. Surface plasmon resonance confirmed high affinity of ALc-B8 and ALc-H7
To examine the basis of the potent inhibition of BoNT/A-Lc protease activity by VHH ALc-B8 and ALc-H7, we measured the solution affinity of Lc protease for VHH-immobilized surface-by-surface plasmon resonance. For these experiments, VHHs were purified following expression and secretion from the E. coli periplasm or expression in the cytosol without noteworthy differences in affinity. As shown in , the affinity (KD) of ALc-B8 to BoNT/A Lc was estimated to be 1.06 nM with an association rate, kon = 6.95 × 104 M−1 s−1 and a dissociation rate, koff = 7.4 × 10−5 s−1. The kinetics of the ALc-H7 binding to A-Lc was comparable as ALc-B8 (KD = 0.66 nM, kon = 7.84 × 104 M−1 s−1, koff = 5.22 × 10−5). The binding of these VHHs to BoNT/A-Lc appeared specific as there was no binding to BoNT/B-Lc. The extremely slow dissociation rate of ALc-B8 and ALc-H7 from BoNT/A found through the affinity studies likely explains the near stoichiometric inhibition of BoNT/A protease by these VHHs.
Fig. 3 Ultra-high affinity binding of VHH-B8 (A) and VHH-H7 (B) to A-Lc measured by surface plasmon resonance. A-Lc was injected over a B8 or H7 coated surface at a series of 2-fold dilutions beginning at 50 nM with the lowest response obtained at 0 nM. The (more ...)
3.5. Anti-BoNT Lc VHHs are functional expressed in neuronal cells
The coding DNA for VHH ALc-B8, ALc-D4 and BLc-B10 were inserted into a mammalian expression plasmid, fused in frame to an amino terminal YFP. The ALc-B8 expression plasmid (YFP/B8) or a control plasmid (pcDNA3.1) was co-transfected into Neuro2a neuroblastoma cells together with an expression plasmid for A-Lc fused to CFP (CFP/A-Lc). A day later the cells were lysed and extracts incubated with a FRET-based assay substrate for BoNT/A protease. Cell extracts containing ALc-B8 were strongly inhibitory of A-Lc proteolytic activity as compared to the control extracts (not shown).
YFP/B8 and YFP/B10 were next tested for their BoNT Lc binding function following expression within Neuro2a neuroblastoma cells. Each of the VHH fusion proteins also contains a streptavidin binding peptide at the amino terminus to permit their rapid purification from cell extracts. The YFP/B8 or YFP/B10 fusion proteins were co-expressed in Neuro2a cells with either CFP/A-Lc or CFP/B-Lc. The next day, the VHHs were purified by streptavidin affinity and the co-purification of BoNT Lc was assessed by Western blot (). CFP/A-Lc was observed in the streptavidin bound fraction when co-expressed with YFP/B8 but not when co-expressed with YFP/B10 indicating that the YFP/B8 was associated with A-Lc in the neuronal cell extracts. In a similar streptavidin pull-down experiment, CFP/B-Lc was found associated with YFP/B10 in the streptavidin bound fraction, but not when co-expressed with YFP/B8, showing that YFP/B10 retains B-Lc binding function when expressed in neuronal cells. The amount of Lc that co-purifies with each VHH appears not to exceed the amount of VHH as expected for a 1:1 stoichiometric interaction. For this reason, some of the targeted Lc remains in the unbound fraction.
Fig. 4 VHH ALc-B8 and BLc-B10 specifically bind to their target Lc expressed in neuronal cell extracts. Neuro2a cells were co-transfected with expression plasmids either for ALc-B8 or BLc-B10 and for BoNT/A-Lc or BoNT/B-Lc as indicated. The Lcs were expressed (more ...)
We next tested whether the BoNT/A-Lc binding VHHs would co-localize with the A-Lc within living neuronal cells using a co-localization strategy. It has been reported that BoNT/A Lc, but not BoNT/B Lc, localizes to plasma membranes when expressed within neuronal cells (Fernandez-Salas et al., 2004a
). As expected, several days following transfection of Neuro2a cells with an expression vector for CFP/A-Lc, CFP fluorescence becomes localized primarily to the plasma membrane (). Transfection of Neuro2a cells with expression plasmids for VHH ALc-B8 (YFP/B8) or ALc-D4 (YFP/D4) in the absence of BoNT/A-Lc results in YFP fluorescence throughout the cell suggesting cytosolic localization. In contrast, when both the A-Lc and the VHHs were co-expressed in Neuro2a, both the CFP and the YFP fluorescence became localized to the plasma membrane after about four days indicating that the VHHs became bound to the A-Lc and co-localized with this membrane-associated protein. These results indicate that the A-Lc binding property remains functional for both A-Lc targeting VHHs, ALc-B8 and ALc-D4, even when they are expressed in the cytosol of neuronal cells.
Fig. 5 VHHs ALc-B8 and ALc-D4 co-localize with BoNT/A protease when co-expressed within cultured neuronal cells. All images are of M17 neuroblastoma cells transfected with one or two expression plasmids as indicated. After four days post-transfection the cells (more ...)
3.6. VHH ALc-B8 inhibits SNAP25 proteolysis following BoNT intoxication of neuronal cells
VHH ALc-B8 is a potent inhibitor of A-Lc and remains fully functional as a binding protein expressed within neuronal cells (see above). To determine whether the VHH functions as an inhibitor of A-Lc when the protease is delivered to neurons by BoNT/A holotoxin intoxication, M17 neuroblastoma cells were transfected with an “indicator” expression plasmid for SNAP25 flanked by an amino terminal YFP and a carboxyl terminal CFP. When these cells were intoxicated with BoNT/A, the indicator protein was cleaved and the level of full-size protein substantially diminished (). When an expression plasmid for VHH ALc-B8 was co-transfected with the plasmid expressing the indicator protein, BoNT/A cleavage of the indicator protein was not detectable, indicating that the A-Lc was inhibited by the VHH.
Fig. 6 VHH ALc-B8 inhibits BoNT/A protease when co-expressed within cultured neuronal cells. (A) M17 cells were co-transfected with an expression vector encoding the indicator protein, YFP/SNAP25/CFP, and either a control plasmid (−) or an expression (more ...)
We next tested whether VHH ALc-B8 could protect the endogenous SNAP25 substrate of A-Lc from cleavage by BoNT/A. This is complicated by the fact that only a portion of cells become transfected with the expression plasmid or become intoxicated by BoNT/A. As such, we can expect to see inhibition of BoNT/A only in the overlapping portion of cells that become both intoxicated and transfected. Thus, these studies have substantial background and multiple replicates were performed. As seen in , cell populations transfected with ALc-B8 expression plasmid have reduced levels of SNAP25 cleavage following intoxication with BoNT/A as compared to cells transfected controls. In this experiment, which included additional replicates, the reduction in cleavage was highly significant (P < 0.01) compared to cells transfected with a control plasmid. Other experiments performed similarly with ALc-B8 transfection into neuroblastoma cells have consistently reproduced this finding (not shown).