Animal immunization
A cynomolgus macaque (Macaca fascicularis) (6 kg) was subcutaneously injected with the non toxic A-chain of ricin (RCA-A) purchased from Sigma, Saint Louis, Missouri. The first injection was RCA-A (100 μg per injection) mixed with complete Freud's adjuvant (Sigma) and then RCA-A mixed with incomplete Freud adjuvant for the remaining injections.
Animal housing was performed in facilities accredited by veterinary services. Animals were provided with NHP feed, water ad libitum, and maintained on a 12-h light cycle. The protocol was approved by the CRSSA ethical committee for animal care and use.
Construction and screening of the anti-ricin antibody gene library
Bone marrow (5 ml) was sampled several times after the last boost in order to isolate RNA using Tri Reagent (Molecular Research Center Inc., Cincinnati, USA) according to the manufacturer's instructions. RNA was retro-amplified, then primers intended for the amplification of DNA encoding macaque Fd and VLκ were utilised as formerly exposed [22]. Later, a set of human λ primers (table 1) was utilised under the same PCR conditions.
PCR products were first cloned in the pGemT vector (Promega, Madison, Wiscontin), according to the manufacturer's instructions, yielding three antibody gene sub-libraries encoding the heavy (Fd fragment) or light (κ or λ) chains. The pGemT cloned PCR products were reamplified using two oligucleotide primer sets for introducing restriction sites for library cloning. A set of macaque κ oligonucleotide primers [21] or of human λ oligonucleotide primer (table 1) were used as forward (annealing to the 5' end of VH or VL) oligonucleotide primers. Only macaque-specific oligonucleotide primers were used as reverse oligonucleotide primers ([21] and table 1). The secondary PCRs were carried with separated forward oligonucleotide primers in order to maintain diversity. Each PCR was performed in 100 μl using 100 ng purified PCR reaction of the pGemT cloned cDNA, 4 U Red Taq (Sigma, Hamburg, Germany), 200 μM dNTPs each and 200 nM of each oligonucleotide primer for 20 cycles (30 s 94°C, 30 s 57°C, 30 s 72°C), followed by 10 min 72°C. The PCR products were separated by a 1.5% (w/v) agarose gel, then cut out and purified using Nucleospin Extract II Kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's instructions.
Construction of the library was performed in two steps. First, the VLκ or VLλ PCR products were cloned to pHAL14 [21, 28, 29] and second, the VH PCR products were cloned to pHAL14 containing the VLκ or VLλ repertoire. The pHAL14 (5 μg), as well 2 μg VL (VLκ or VLλ), were digested using 50 U MluI and 50 U NotI (NEB, Frankfurt, Germany) in a 100 μl reaction volume (2 h at 37°C). The digest was inactivated by heating at 65°C for 10 min. Afterwards, 0.5 U calf intestinal phosphatase (MBI Fermentas) was added to the digested pHAL14 and incubated for an additional 30 min. This step was repeated once. The vector was purified using the Nucleospin Extract II Kit and 270 ng VL were cloned into 1 μg of the dephosporylated pHAL14 by incubating overnight (16°C) with 1 U ligase (Promega, Mannheim, Germany). The ligation reactions were precipitated with ethanol plus sodium acetate, and the pellet washed two times with 70% ethanol. These reactions were electroporated (1.7 kV) in 25 μl XL1-Blue MRF' (Stratagene, Amsterdam, Netherlands). The transformed bacteria were plated onto SOB agar plates (25 cm petri dishes) supplemented with 100 μg/mL ampicillin and 100 mM glucose. These colonies were harvested by suspending in 40 mL SOB media. Plasmids from the VLκ or VLλ library were isolated using the Nucleobond Plasmid Midi Kit (Macherey-Nagel) according to the manufacturer's instructions. Five micrograms of each VL chain library, as well 1.5 μg of the VH fragments, were digested using 50 U NcoI and 100 U HindIII (NEB) in a 100 μl reaction volume (overnight at 37°C). The following steps were performed as described for VL with the following modification: 250 ng of the digested and purified VH repertoire was used for ligation. In total 2 transformations were performed. The harvested bacteria, containing the final antibody gene libraries, were aliquoted and stored at -80°C.
For library packaging, 500 mL 2xTY medium [30] containing 100 μg/mL ampicillin and 100 mM glucose were inoculated with a 1 mL aliquot of the antibody gene library. Bacteria were grown (37°C, 250 rpm rotary shaker incubator) to an O.D.600 of 0.4 – 0.5. Twenty-five millilitres of bacteria (~1,25 × 1010) were infected with 2,5 × 1011 Hyperphage [31–33], incubated at 37°C for 30 min without shaking, followed by 30 min at 250 rpm. The infected cells were harvested by centrifugation (10 min, 3,220 × g). The pellet was resuspended in 200 mL 2xTY containing 100 μg/mL ampicillin and 50 μg/mL kanamycin. The phages were produced at 30°C and 250 rpm for 16 h. Cells were pelleted by centrifugation for 10 min (10,000 × g). The supernatant containing the phages was precipitated with 1/5 volume of 20% (w/v) polyethylene glycol (PEG)/2.5 M NaCl solution (1 h on ice) with gentle shaking and then pelleted by centrifugation for 1 h at 10,000 × g (4°C). The precipitated phage were resuspended in 10 mL phage dilution buffer (10 mM TrisHCl pH7.5, 20 mM NaCl, 2 mM EDTA) and filtered through a 0.45 μM filter. Phage precipitation was repeated once more. The phage pellet was diluted in 1 mL phage dilution buffer and cell debris was pelleted by additional centrifugation (5 min. at 15,400 × g, 20°C). The supernatant containing the phage were stored at 4°C. Each library packaging was controlled by tittering, and subsequently tested by immunoblot according to [28, 34].
Screening of the library was performed as described elsewhere [35, 36], except that 10, 15, and 30 washes were performed for each successive round of panning. Ricin was utilized as the antigen and PBS-Tween 20 0.1% as the washing buffer.
scFv production, ELISA testing
Phagemid DNA isolated after the panning process was used to transform the non-suppressor E. coli strain HB2151 such that it expressed the soluble scFv fragment. Single colonies of randomly chosen transformants were used to inoculate 5 ml of SB (Super Broth) medium supplemented with carbenicillin (50 μg.ml-1) and 1% glucose. In parallel (see below), the clones were sequenced so that redundant clones were not re-analyzed. For expression, cultures were incubated overnight (30°C) with vigorous shaking (250 rpm). Five hundred milliliters of SB medium supplemented with carbenicillin and 0.1% glucose were then inoculated with 500 μl of each culture. The cultures were grown at 30°C until the 600 nm optical density (OD) reached a value of one. In order to induce gene expression, IPTG (1 mM) was added and the cultures incubated overnight (22°C). The cells were harvested by centrifugation at 2500 × g for 15 min, 4°C. The scFvs were extracted with polymyxin B sulphate [37] and purified using a nickel column (Ni-NTA spin column, Qiagen, Valencia, CA) according to the manufacturer's instructions.
For ELISAs, RCA-A or ricin (Sigma, 10 μg/ml PBS) was coated onto a 96 well microtitre plate (Maxisorp, Nunc, Danemark). When sera were tested, the negative control was pre-immune serum and secondary antibody reporter utilized polyclonal anti-macaque IgG, Fc specific tagged with HRP (Nordimmune, Tilburg, The Netherlands). The antibody titre was measured as the reciprocal of the highest dilution of immune serum giving a signal three times stronger than the negative control, at the same dilution. When scFv were tested, they were detected by an anti-his tag antibody (Qiagen, Courtaboeuf, France).
Ricin neutralization in cell-based assays
Soluble scFvs, eluted after the library panning and expressed individually, were tested for their neutralization capacity using a cell-based viability assay. In this assay, cells are put in contact with ricin, in such conditions that all cells die. The scFv to be tested is added, at different concentrations, to ricin and may inhibit its toxic activity. At the end of the assay, cell viability is assessed and plotted against scFv concentration. This assay replicates the two main steps of ricin inhibition (internalization of the scFv bound to ricin and inhibition of its toxic activity). More precisely here, J774A.1 cells (ATCC-LGC, Molsheim, France) were plated at a density of 14,000 cells/well (200 μl/well), cultured (37°C with 5% CO2) for 24 hours in DMEM supplemented with 10% SVF. Ricin, at a concentration of 15 ng/ml (corresponding to 10 LD50, data not shown), was pre-incubated with scFvs, or with control serum for 1 h (37°C) and then added to the cells. After a 24 h incubation (37°C, 5% CO2), cell viability was determined by using the Cytotox 96 Non-radioactive Cytotoxicity Assay (Promega, Madison, Wi), as suggested by the manufacturer. Each assay was corrected for 100% cell viability (control wells with no toxin and no scFv) and 0% viability (control wells with ricin and no scFv). The scFv concentration corresponding to 50% viability is defined as the IC50 (inhibitory concentration 50%). These assays were performed three times in triplicate. All experiments were conducted on J774A.1 cells sub-cultured less than 15 times after receiving them from the vendor.
ScFv stability
The scFv stability was estimated by determining the percentage of scFv active after a 24 h incubation at 37°C in DMEM medium. ScFvs (50 μg/ml in PBS) were incubated in triplicate and then tested in an ELISA, utilizing a freshly thawed aliquot for control.
Ricin neutralization in a cell-free translation assay
In this assay, a cell-free translation system is put in contact with ricin, resulting in translation inhibition. The scFv is added, at different concentrations, to ricin and may inhibit its toxic activity. At the end of the assay, translation activity is plotted against scFv concentration. This assay replicates the intra-cellular step of ricin inhibition. More precisely here, the neutralizing activity of the best scFv was determined using a microtitre cell-free translation assay that detects luciferase translation from luciferase m-RNA [38]. By using phosphate buffered saline (PBS), antibodies were diluted in a 96 well microtitre plate and a constant amount of ricin (4 nM final concentration) was added to the antibody dilutions. Dilutions of ricin alone were included in each assay for generation of a standard. In addition, anti-ricin IgGs, purified from serum of mice that had been hyperimmunized with the dgRCA-A to result in antibodies with a high neutralization activity of ricin, were utilized as a benchmark of neutralization. The plate was placed on a microplate shaker for 15 min (25°C), and then 5 μl transferred to a v-shaped microtitre plate. The rabbit reticulocyte lysate, RNasin, amino acids complete, and luciferase m-RNA (Promega, Madison, WI) were mixed together and 25 μl added to each well of the v-shaped plate. Plates were incubated (37°C) for 90 min. Five microliters of the translation lysate were transferred to a black microtitre plate (Sigma Chemicals, St. Louis, MO). After the addition of 45 μl reaction buffer (Luciferase assay reagent, Promega, Inc.), luminescence was measured as counts per second (CPS) on a Victor multiplate reader (PerkinElmer Wallac, Boston MA). Data was expressed as the percent of control [% control = (CPS treated/CPS control) × 100].
Affinity measurements
Affinities were measured by surface plasmon resonance (SPR) with a BIAcore™ × (Biacore, Uppsala, Sweden) instrument. The scFv 43RCA was immobilized at a maximum of 100 RU on a CM5 chip (Biacore) via amine coupling, according to the manufacturer's instructions. A 30 μl/min flow rate was maintained during measurement. For each measurement, eight ricin dilutions were prepared in HBS-EP buffer (Biacore) (concentrations ranging from 1 to 250 nM) and were tested at various elution times from 600 sec to 14,500 sec. For the precise measurement of very high affinities, we were advised (C. Quetard, Biacore, France) to use long elution times with the highest tested ricin concentration. After each ricin dilution, the chip was regenerated with 1.5 M glycine buffer (Biacore), run 10 μl/min for 30 seconds. Constants were calculated [39], and verified with internal consistency tests [40] as formerly described.
Nucleic acid analysis of ricin-specific scFv clones
In parallel to expression, transformed HB2151 bacteria were cultivated to isolate the phagemidic DNA (Nucleobond AX, Macherey-Nagel). Variable region sequences of the light and heavy chains were determined by Genome Express (Meylan, France) using the primers Mkmyc and MkpelB, respectively [41]. The sequences were analyzed on line, using the International ImMunoGeneTics information system ® (IMGT) [42]http://imgt.cines.fr and its nomenclature. In particular, these sequences were compared with those of the human germline immunoglobulin genes by using the IMGT/V-QUEST [43] and IMGT/JunctionAnalysis softwares [44]. Peptidic sequence comparisons utilized new IMGT tools [26], which classify and compare three separate items; hydropathy, volume, and chemical properties, for each amino acid. This three item analysis was summarized as follows: when no significant difference between two residues was recognised in the three items, residues were regarded as highly similar. When one difference was found, the residues were regarded as similar but then regarded as dissimilar in the case of two differences, and very dissimilar in the case of three differences.
In addition, the similarities between all analysed scFv sequences were analysed using a new on-line tool http://www.phylogeny.fr/, which builds phylogenetic trees. Phylogenetics in the strict sense apply to immunoglobulins because, due to somatic hypermutations that are the support for affinity maturation, unmutated rearranged germline sequences are ancestors of those coding for high affinity antibodies. After library panning however, only high affinity, mutated antibody fragments are retained. Thus, in this study, we utilised this tool as a multiple alignment software that displays results in the form of phylogenetic trees or sub-trees, and applied it to the sequences coding scFvs selected from the library. In such trees, each node, where certain branches of a tree meet, indicates the consensus shared by the different sequences displayed at the extremity of corresponding branches. The length of each branch, between each node and each sequence, is in proportion with the differences between the corresponding consensus and sequence.
Nucleotide sequences
The Macaca fascicularis 43RCA-H and 43RCA-L sequences (VH and VL domain sequences, respectively) are accessible in the data banks under the accession numbers [EMBL: FJ178346 and EMBL: FJ178347].