Cells and viruses
BHK-21, Vero and C6/36 Aedes albopictus
cells were cultured as previously described 35
. J774.2 mouse macrophages and SW13 human adrenal cortex adenocarcinoma cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). The majority of experiments were performed with the WNV strain (3000.0259, passage 2) that was isolated in New York in 2000. Additional neutralization experiments were performed with lineage I strains from New York from the years 2000 to 2003 (03001956, 32010157, NYC01035, 03002094, 02002640, 02002831, 02003688, 31000352, 00–7365) and a lineage II strain (956) isolated from Uganda in 1937 24,36
. Neutralization experiments were also performed with prototype strains of St. Louis (59268 (Parton)) and Japanese encephalitis (Nakayama) viruses 37
. For in vivo
experiments, viruses were diluted and injected into mice as described 23
Purified WNV E protein expression
WNV E protein ectodomain was generated using a baculovirus expression system according to previously described methods for related flaviviruses 38
. The last 45 nucleotides of prM (endogenous signal sequence) and the first 1290 nucleotides of WNV E protein from the New York 1999 strain 39
were fused downstream of the polyhedrin promoter and upstream of a histidine repeat in a baculovirus shuttle vector (pFastBac, Invitrogen, Carlsbad, CA) by PCR using a high-fidelity Taq polymerase (Platinum Taq, Invitrogen). Three days after baculovirus infection of Hi-5 insect cells at a multiplicity of infection (MOI) of 1, supernatants were harvested, filtered, buffer-exchanged and purified by nickel-affinity chromatography according to the manufacturer’s instructions. The purified WNV E ectodomain lacks the C-terminal 71 amino acids that are associated with the membrane proximal, transmembrane, and cytoplasmic domains.
Purified WNV DIII
The construction, expression, purification, and refolding of DIII of WNV E protein is described in greater detail elsewhere (G. Nybakken, T. Oliphant, M. Diamond, and D. Fremont, manuscript submitted). Briefly, wild type, N394K, and K307N DIII were generated from an infectious cDNA clone of the New York 1999 strain of WNV (gift of R. Kinney, Fort Collins, CO) using PCR and Quik-change mutagenesis (Stratagene, La Jolla, CA). After cloning into a PET21 vector (Novagen, San Diego, CA) and sequence confirmation of the mutations, plasmids were transformed into BL21 Codon Plus E. Coli cells (Stratagene). Bacteria were grown in LB, induced with 0.5 mM isopropyl thiogalactoside (IPTG), and pelleted. Subsequently, bacteria were lysed after the addition of lysozyme, sonicated, and DIII was recovered as insoluble aggregate from the inclusion bodies. DIII was denatured in the presence guanidine hydrochloride and β-mercaptoethanol and refolded by slowly diluting out the denaturing reagents in the presence of L-arginine, EDTA, PMSF, reduced glutathione, and oxidized glutathione. Refolded DIII was separated from aggregates on a Superdex 75 16/60 size exclusion column (Amersham Bioscience), concentrated using a centricon-10 spin column into 20 mM Hepes pH 7.4, 150 mM NaCl, and 0.01% NaN3. After refolding, wild type DIII reacted with all domain-III specific mAbs including those that recognized conformationally sensitive epitopes.
MAb generation and purification
BALB/c mice were primed and boosted at three-week intervals with insect cell-generated, purified, recombinant WNV E protein (25 μg) that was complexed with adjuvant (RIBI Immunochemical, Hamilton, MT). Approximately one month after the last boost, serum was harvested and tested for immunoreactivity against solid-phase purified E. Mice with high titers (> 1/10,000) were boosted intravenously with purified E protein (5 μg) in PBS. Three days later, splenocytes were harvested and fused to P3X63Ag8.653 myeloma cells to generate hybridomas according to published procedures 40
. MAbs against WNV or other control antigens were purified by standard protein A or protein G chromatography according to the manufacturer’s instructions (Pharmacia, Piscataway, NJ).
For WNV infection experiments, all wild type C57BL/6J mice were derived were purchased from a commercial source (Jackson Laboratories, Bar Harbor, ME). The congenic C1q-deficient and C4-deficient mice were obtained from Gregory Stahl (Boston, MA) and Michael Carroll (Boston, MA), respectively. The congenic Fc γ R I and III-deficient mice were obtained commercially (Taconic, Germantown, NY). Mice were used between 5 and 8 weeks of age depending on the particular experiment and inoculated subcutaneously with WNV by footpad injection after anaesthetization with xylazine and ketamine. Mouse experiments were approved and performed according to the guidelines of the Washington University School of Medicine Animal Safety Committee.
For passive transfer experiments, mice were administered a single dose of purified mAb by intraperitoneal injection at a given time point (day 2, 4, or 5) after infection. To analyze virus production in the brain, infected mice were euthanized on a given day after inoculation. After cardiac perfusion with PBS, brains were removed, weighed, and homogenized, and plaque assays were performed as previously described 23
Expression of WNV E protein on yeast
The ectodomain or DIII of WNV E protein was expressed on the surface of yeast using a modification of a previously described protocol for surface expression of T cell receptors 29
. Amino acid residues 1–415 (ectodomain) or 296–415 (DIII) of WNV E protein were amplified with BamHI and Xho I sites at their 5′ and 3′, respectively, by PCR from the New York 1999 infectious cDNA clone (R. Kinney, Fort Collins, CO). The resulting products were digested with BamHI and XhoI, and cloned as downstream fusions to the yeast Aga2 and Xpress™
epitope tag genes in the yeast surface display vector pYD1 (Invitrogen). An upstream GAL1 promoter controls fusion protein expression. These constructs were transfected into the S. cerevesiae
yeast strain EBY100 25,41
resulting in yeast that expressed the WNV E ectodomain or DIII. Yeast that only expressed the Xpress™
epitope tag linked to Aga2 were prepared in parallel by transfecting EBY100 cells with the parent vector pYD1. Individual yeast colonies were grown to log phase overnight in Trp−
media containing 2% glucose at 30° C and harvested in log phase. Fusion protein expression was induced on the yeast surface by growing yeast for an additional 24 hours in Trp−
media containing 2% galactose at 25° C. Yeast were harvested, washed with PBS supplemented with 1 mg/ml BSA and immunostained with 50 μl of mAb (25 μg/ml) against the Xpress™ tag or WNV E protein. After 30 minutes, yeast were washed thrice and stained with a goat anti-mouse secondary antibody conjugated to Alexa Flour 647 (Molecular Probes, Eugene, OR). Subsequently, the yeast cells were analyzed on a Becton Dickinson FACSCaliber flow cytometer.
Library construction and screening
DIII of the WNV E protein was mutated using an error-prone PCR protocol 25
that included Mn2+
at concentrations of 0.3 and 2.0 mM. Subsequently, the cDNA library was ligated into pYD1 and transformed into XL2-blue ultracompetent cells (Strategene, La Jolla, CA). The colonies were pooled and the plasmid DNA was recovered using the Qiagen HiSpeed Maxi kit.
For each individual antibody, the yeast library of DIII mutants was screened according to the following protocol. To identify yeast that selectively lost binding to a given mAb epitope, the library was initially stained with an Alexa Flour 647-conjugated anti-WNV mAb for 30 minutes at 4°C. To control for the surface expression of DIII, after washing, yeast were subsequently stained for 30 minutes at 4°C with an Alexa Fluor 488-conjugated oligoclonal antibody that was derived from a pool of individual mAb antibodies (E1, E2, E9, E16, E24, and E34). After immunostaining, yeast were subjected to flow cytometry and the population that was single mAb negative but pooled oligoclonal antibody (oligoAb) positive was identified. The yeast cells were sorted at an event rate of ~4000 cells per second and this population (mAb− and oligoAb+) was enriched after three rounds of sorting. After the final enrichment sort, yeast were plated and individual colonies were selected and tested for binding to individual mAbs. For individual clones that had lost only the desired mAb epitope, the DIII-pYD1 plasmid was recovered using the Zymoprep Yeast Miniprep kit (Zymo Research, Orange, CA). The plasmid was then transformed into DH5α cells, purified using the Qiaprep Spin Miniprep kit (Qiagen, Valencia, CA) and sequenced.
In some cases, DIII variants with two independent mutations were isolated. To determine which mutation conferred the loss-of-binding phenotype, single independent mutations were engineered by site-directed mutagenesis of DIII-pYD1 using mutant oligonucleotides and the Quik Change II mutagenesis kit (Strategene). All mutations were confirmed by sequencing.
Quantitation and characterization of neutralizing antibodies in vitro
The titer of neutralizing antibodies was determined by a standard plaque reduction neutralization titer (PRNT) assay using either BHK21 or SW13 cells 23
. Results were plotted and the titer for 50% (PRNT50
) and 90% inhibition (PRNT90
) was calculated. The inhibition assay with J774.2 mouse macrophages was performed as follows: Medium, E16 or E24 (2.5 μg of mAb) was mixed with 5 × 102
PFU of WNV, incubated for 1 h at 4°C, and then added to 5 × 104
J774.2 mouse macrophages in individual wells of a 24 well plate. After 1 h, cells were washed four times with PBS to remove free virus and mAb, DMEM with 10% FBpS was added, and the cells were incubated for an additional 24 hours. Supernatants were subsequently harvested for a viral plaque assay on Vero cells.
Competition ELISA with human anti-WNV antibodies
After purification and refolding, wild type, K307N, and N394K DIII were diluted (5 μg/ml) in 0.1 M Na carbonate buffer (pH 9.3) and adsorbed to 96-well plates overnight at 4°C. After blocking with PBS, 2% BSA and 0.05% Tween 20 (PBS-BT), wells were pre-incubated for one hour at room temperature with PBS-BT containing no antibody, E16 IgG (50 μg/ml), E16 Fab (50 μg/ml) or E53 IgG (50 μg/ml). E53 serves as a negative control as it recognizes an epitope in domain I and II of WNV E protein. Subsequently, human plasma (1/40 dilution in PBS-BT, heat-inactivated) was directly added for an additional hour at room temperature. The human samples were obtained with informed consent from seven different WNV-infected patients (gift of M. Busch and L. Tobler, San Francisco, CA), Because the samples were sequentially numbered and not linkable back to the original subjects, they satisfied the criteria for exemption from approval from the Human Studies Committee at Washington University. After 6 washes with PBS-BT, plates were serially incubated with biotin-conjugated goat anti-human IgG (1 μg/ml), streptavidin-horseradish peroxidase (2 μg/ml) and tetramethylbenzidine developing substrate (DAKO, Carpinteria, CA), Optical densities at 450 nm were determined with an automatic ELISA plate reader (Tecan, Research Triangle Park, NC) and adjusted after subtraction of the value obtained from non-immune human plasma.
Surface Plasmon Resonance
Antibody affinity for DIII of WNV was performed by surface plasmon resonance (BIAcore 3000, Biacore, Inc, Neuchatel, Switzerland). Binding curves and kinetic parameters were obtained as follows: E16 antibodies were captured by flowing (300 nM, rate of 5 ml/min for 2 minutes) them over immobilized F(ab)′2 fragment that was specific for Goat anti-human IgG with Fc region specificity. Subsequently, DIII of the New York 1999 strain of WNV E protein (amino acids 296 to 415), which was generated in E Coli (G. Nybakken, T. Oliphant, M. Diamond, and D. Fremont, manuscript submitted), was injected (6.25–200 nM, flow rate 70 ml/min for 1.5 minutes and then allowed to dissociate over 5 minutes). The F(ab)′2 surface was regenerated by pulse injection of 10 mM Glycine pH 1.5 and 100 mM NaOH before each E16 injection. Curves were analyzed with a Global fit 1:1 binding algorithm with drifting baseline.
Cloning and humanization of E16
E16 heavy and light chain RNA was isolated from hybridoma cells after guanidinium thiocyanate and phenol-chloroform extraction, and converted to cDNA by reverse transcription. The VH and VL segments were amplified by PCR using the 5′ RACE system (Invitrogen). Gene specific primers (GSP) for VH and VL were as follows: VH-GSP1: 5′-GGTCACTGTCACTGGCTCAGGG-3′; VH-GSP2: 5′-AGGCGGATCCAGGGGCCAGTGGATAGAC-3′; VL-GSP1: 5′-GCACACGACTGAGGCACCTCCAGATG-3′; and VL-GSP2: 5′ CGGATCCGATGGATACAGTTGGTGCAGCATC-3′. The RACE products were inserted into the plasmid pCR2.1-TOPO using the TopoTA kit (Invitrogen). The resulting plasmids were then subjected to DNA sequencing to determine the VH and VL sequences for E16. The cDNA sequences were translated and the predicted amino acid sequence determined. From these sequences the framework (FR) and CDR regions were identified as defined by Kabat 42
. The mouse VH was joined to a human C-γ1 constant region and an Ig leader sequence, and inserted into pCI-neo for mammalian expression. The mouse VL was joined to a human Cκ segment and an Ig leader sequence and also cloned into pCI-neo for mammalian expression of chimeric E16 (Ch-E16). For Ch-E16, site-directed mutagenesis was also performed to change residue 297 from asparagine to glutamine of the heavy chain to eliminate the single glycosylation site on the γ1 Fc.
Humanized E16 VH consists of the FR segments from the human germline VH1-18 VH segment and JH6 segment 43,44
, and the CDR regions of the E16 VH, respectively. The humanized E16 VL consists of the FR segments of the human germline VK-B3 VL segment and JK2,02 45–47
segment and the CDR regions of E16 VL. The humanized VH segments were assembled de novo
from oligonucleotides and amplified by PCR. The humanized VL segments were assembled by PCR and overlapping PCR. The resulting VH and VL segments were subsequently combined by overlapping PCR with a leader sequence and the appropriate constant region segment and cloned into the expression vector pCI-neo as Nhe I–EcoR I fragments. The DNA sequence of the resulting plasmids was confirmed by sequence analysis. Site-directed mutagenesis was then performed to substitute mouse for human residues at key framework positions VH-71 (T71A) and VL-49 (Y49S). The resulting plasmids were co-transfected into human 293 cells using lipofectamine-2000 and humanized antibody was recovered from the resulting conditioned medium and purified by protein A and size exclusion chromatography.
All data were analyzed with Prism software (GraphPad Software, San Diego, CA). For survival analysis, Kaplan-Meier survival curves were were analyzed by the Logrank and Mantel-Haenszel test. For viral burden and experiments, statistical significance was determined using the Mann-Whitney test.