Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Nat Med. Author manuscript; available in PMC 2006 May 9.
Published in final edited form as:
Published online 2005 April 24. doi:  10.1038/nm1240
PMCID: PMC1458527

Development of a Humanized Monoclonal Antibody with Therapeutic Potential against West Nile Virus


Neutralization of West Nile virus (WNV) in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Using random mutagenesis and yeast surface display, we defined individual contact residues of 14 newly generated mAbs against domain III of the WNV E protein. MAbs that strongly neutralized WNV localized to a surface patch on the lateral face of domain III. Convalescent antibodies from human patients who had recovered from WNV infection also detected this epitope. One mAb, E16, neutralized 10 different strains in vitro, and demonstrated therapeutic efficacy in mice, even when administered as a single dose 5 d after infection. A humanized version of E16 was generated that retained antigen specificity, avidity, and neutralizing activity. In post-exposure therapeutic trials in mice, a single dose of humanized E16 protected mice against WNV-induced mortality, and thus, may be a viable treatment option against WNV infection in humans.

WNV is a single-stranded, positive-polarity RNA Flavivirus that is related to dengue fever, yellow fever, and Saint Louis, tick-borne, and Japanese encephalitis viruses. Humans infected with WNV develop a febrile illness that can progress to meningitis or encephalitis, and the elderly and immunocompromised are at greatest risk for severe disease 1. At present, treatment is supportive and no vaccine exists for human use.

The innate and adaptive immune responses prevent dissemination to and within the central nervous system (CNS) 2,3. Recently, two groups demonstrated therapeutic efficacy of immune human γ-globulin in mice infected with WNV 4,5. Even after virus had spread to the CNS, passive administration of immune heterologous γ-globulin improved survival 5. In theory, a potently neutralizing mAb could have the same or better benefit with a lower dose and improved safety profile.

Most neutralizing antibodies against flaviviruses recognize the envelope (E) protein. In general, virus-specific rather than cross-reactive antibodies have the strongest neutralizing activity in vitro and greatest protection in vivo 6. Crystallographic analysis of the soluble ectodomain of flavivirus E proteins has revealed three domains 7,8. Domain I is an 8-stranded β-barrel 79 that participates in the conformational changes associated with the acidification in the endosome 10. Domain II contains 12 β-strands and has roles in dimerization, trimerization, and fusion 7,8,10. Domain III (DIII) adopts an immunoglobulin-like fold, and contains the loops that are most distal from the surface in the mature virion 11,12 and the site for putative receptor attachment 6,8,13,14. Based on the sequencing of in vitro neutralization escape variants, many neutralizing antibodies against flaviviruses localize to DIII 1522.

Here, we define further the molecular basis of antibody-mediated neutralization of WNV using a large panel of newly generated mAbs against WNV E protein. Humanized versions of one of these, E16, retained antigen specificity, avidity, neutralizing activity, and protected mice against WNV-induced mortality.


Post-exposure treatment with neutralizing polyclonal human γ-globulin partially protects mice against WNV 5. While human γ-globulin has potential as an immunotherapy for WNV infection, it has several limitations: (a) it is derived from non-immune and immune donors and has only a modest specific neutralizing titer 5; (b) batch variability may affect the efficacy of specific preparations; and (c) as a human blood product, it has an inherent risk of transmitting infectious agents. To overcome these limitations, we developed a panel of mouse mAbs against WNV and determined the in vitro and in vivo inhibitory potency as a guide for identifying candidates for humanization.

Generation of mAbs against WNV E protein

We fused the first 1,290 nucleotides of WNV E protein upstream of a histidine repeat in a baculovirus shuttle vector. The resultant truncated E protein lacked the C-terminal 71 amino acids that correspond to the transmembrane and cytoplasmic regions. We generated recombinant baculoviruses, infected Hi-5 insect cells, and purified soluble E protein by nickel-affinity chromatography (data not shown). After immunization and screening 2,000 hybridomas, we isolated 46 new mAbs that recognized WNV E protein (Supplementary Table 1).

Neutralizing activity in vitro

We evaluated the mAbs for their ability to block WNV infection in BHK21 cells using a standard plaque reduction assay 23. Twelve had strong neutralizing activity that greatly exceeded the potency of immune human γ-globulin, with 50% plaque reduction neutralization titers (PRNT50) below 2 μg whereas immune human γ-globulin had a PRNT50 value of 500 μg 5. The inhibitory activity of two neutralizing mAbs, E16 and E24, was reproduced in J774.2 mouse macrophages and SW13 human adrenal carcinoma cells (Supplementary Fig. 1) and thus, was not specific to fibroblasts.

One of the potent neutralizing mAbs, E16, inhibited infection of genetically diverse WNV lineage I strains that were isolated from New York. E16 neutralized all WNV strains with PRNT50 values of 4 to 18 ng and PRNT90 values of 53 to 297 ng (Supplementary Table 2). Interestingly, Fab fragments of E16 inhibited WNV (PRNT50; 23 ng), suggesting that neutralization does not require bivalent E protein binding. E16 potently blocked infection with strain 956, the original lineage II strain isolated in 1937 24, yet was virus-specific as it neither recognized nor neutralized other flaviviruses including distantly related dengue and yellow fever viruses (data not shown) and closely related Japanese and St. Louis encephalitis viruses (Supplementary Table 2).

Mapping of neutralizing mAbs to DIII

To map our strongly neutralizing mAbs, we developed a strategy using yeast surface display 25. The ectodomain (amino acids 1–415) or DIII (amino acids 296–415) of WNV E protein were expressed as fusion proteins on the yeast cell surface (Fig. 1a). Most of our 46 mAbs recognized yeast that displayed the entire ectodomain of E (Fig. 1a and Supplementary Table 1). Sixteen mAbs recognized yeast that displayed DIII alone, and 10 of 12 strongly neutralizing mAbs localized to DIII. Only two neutralizing mAbs (E53 and E60) recognized the E ectodomain but not DIII alone.

Figure 1
Mapping of mAbs to DIII with yeast. A. Surface display of WNV E protein on yeast. A fusion protein is composed of the ectodomain or DIII of WNV E protein and the yeast Aga2 protein, which becomes attached to the Aga1 protein in the yeast cell wall. Yeast ...

Mapping of amino acid contact residues of DIII mAbs

We used error-prone PCR mutagenesis of DIII of WNV E protein and yeast surface expression to map antibody contact residues in a high-throughput manner. We performed individual screens to identify DIII mutants that lost binding selectively to strongly neutralizing (E16, E24, and E34), weakly neutralizing (E1), and non-neutralizing (E2 and E22) mAbs. To eliminate mutants that abolished surface expression of DIII, yeast were stained sequentially with an Alexa Fluor 647-conjugated individual mAb and an Alexa Fluor 488-conjugated oligoclonal antibody derived from a pool of individual mAbs. After cell-sorting, we identified yeast that selectively lost expression of an individual mAb epitope but retained surface expression of DIII (Fig. 1b). Multiple independent yeast that lost binding of individual mAbs were subjected to plasmid recovery and sequencing.

MAbs that localized to DIII and strongly neutralized WNV had reduced binding when residues S306, K307, T330 or T332 were altered (Fig. 2a and Table 1). These are located on adjacent loops and form a contiguous patch 16 on the solvent exposed surface at the lateral tip of the DIII (Fig. 2b). Only two other mutations caused significant loss-of-binding of any of the ten neutralizing mAbs tested: K310E or P315R reduced binding only of E49. No two neutralizing mAbs had identical loss-of-binding patterns. For example, S306L reduced binding of E16, E27, E40, E43, and E49 but not E24, E33, E34, E47, and E58. K307R abolished binding of E34, E40, E43, E47, E49, and E58 but affected E16, E24, E27, and E33 less strongly. In contrast, K307E decreased binding of all neutralizing mAbs yet did not affect non- or poorly neutralizing mAbs. Changes in residues T330 and T332 also abolished binding of neutralizing but not non-neutralizing mAbs. T330I or T332M strongly reduced binding of all neutralizing mAbs with the exception of E27, and T332V or T332A weakened binding of only E24, E27, E33 and E58.

Figure 2
Fine epitope mapping of DIII neutralizing and non-neutralizing mAbs. A. Flow cytometry profiles for immunoreactivity by a non-neutralizing (E2), weakly neutralizing (E1) and strongly neutralizing (E34) mAb with yeast expressing wild type and variant DIII: ...
Table 1
Binding of mAbs to domain III variants on the surface of yeast

Six mAbs that recognized DIII were either poorly or non-neutralizing, and none engaged the dominant neutralizing epitope defined by S306, K307, T330 or T332. E2 and E9 were abolished or reduced by mutation of D381 and H396, and binding of E22 was weakened by a change in P315. D381 and H396 are proximal to one another but physically distinct from the four residues that affect binding of neutralizing mAbs (Fig. 2b). None of the mutations identified by loss-of-binding sorts for E2 or E22 had any effect on two other non-neutralizing mAbs, E21 and E23. E1, a mAb with weak neutralizing activity, mapped to a site between the non-neutralizing and neutralizing mAbs, as mutation of K310 and N394 strongly inhibited binding.

Patient antibodies recognize the dominant neutralizing epitope

To determine whether human anti-WNV antibodies recognize the neutralizing epitiope on DIII during infection, plasma was obtained from WNV-positive patients. Convalescent samples were negative for WNV RNA but positive for neutralizing anti-WNV antibodies. The patients reported mild systemic illness although none progressed to severe disease. To determine whether these samples contained antibodies that localized to the neutralizing epitope on DIII, we tested whether E16 Fab or IgG could compete binding to recombinant, wild type and mutant N394K and K307N forms of DIII (Supplementary Fig. 2); N394K retains wild type binding to E16 whereas K307N has markedly reduced binding. E16 equivalently inhibited binding of patient anti-WNV antibodies to wild type (Fab, 35% ± 8; IgG, 40% ± 12) or N394K DIII (Fab, 32% ± 9; IgG, 34% ± 11), whereas E53 IgG, an anti-WNV mAb that recognizes an epitope outside DIII, did not compete binding to wild type (1% ± 4) or N394K (−2% ± 5) DIII. As expected, E16, which only weakly recognizes K307N, poorly competed (Fab 6% ± 3; IgG 9% + 4) binding to K307N DIII. This data suggests that humans, who clear WNV infection, develop antibodies that recognize an epitope in close proximity to that defined by E16.

Therapeutic studies in mice

To evaluate the correlation between neutralization, epitope localization, and in vivo protection, we assessed the therapeutic activity of different neutralizing mAbs in an established mouse model 5. Studies were performed with five week-old wild type C57BL/6 mice, which have a ~10% survival rate 5. Mice were inoculated subcutaneously with 102 PFU of WNV and administered a single dose of mAb at day 2 after infection; Notably, 500 μg of the non-neutralizing mAb E2, provided no protection (data not shown). In contrast, 100 μg of any of three different neutralizing mAbs that map to K307 (E16, E24, or E34) protected greater than 90% of mice from lethal infection (Fig. 3a, b, and c). Even a single 4 μg treatment of E16 or E34 on day 2 after infection prevented mortality.

Figure 3Figure 3
Therapeutic effect of DIII neutralizing mAbs. (A, B, and C) Dose-response analysis at day 2 after WNV infection.. At 2 days after WNV infection, mice were passively transferred a single dose (0.8, 4, 20, 100, or 500 μg) of (A) E16, (B) E24, or ...

Since humans can present with CNS WNV infection, we evaluated the therapeutic efficacy of mAbs at later time points. At days 4, 5 and 6 after WNV infection, we detected virus in the brains of 67%, 78% and 83% of mice (Fig. 3d). A single 500 μg dose of E16 or E34 at day 4 resulted in an 80–90% survival rate (Fig. 3e). A single 2 mg dose of E16 at day 5 resulted in 90% survival (Fig. 3f) and complete clearance of WNV from the brain in 68% of mice by day 9 (Fig. 3g). Thus, administration of neutralizing mAbs to mice with active CNS infection improved survival and induced a virologic cure. As expected, lengthening the interval before treatment was associated with decreased benefit. Administration of E16 at day 6 did not enhance survival (data not shown), although average survival time was increased (9.5 d ± 0.4 to 11.5 d ± 0.4 (P = 0.003)).

Humanization of E16

We considered humanizing E16 or E34 as a possible therapeutic. Humanized mAbs have substantially longer half-lives in humans than their mouse counterparts 26,27. Sequencing studies indicated that E16 had greater homology to human framework regions, making it simpler to construct a humanized version of E16.

(a) Construction and expression of humanized E16

We amplified the cDNA encoding the heavy (VH) and light (VL) variable domains from the hybridoma cellular RNA by a 5′ RACE procedure. The VH belongs to mouse heavy chain subgroup II (J558 family) and the VL belongs to mouse κ chain subgroup V. The complementarity determining regions of E16 were grafted onto the human VH1-18 backbone (Fig. 4a) and human Vκ-B3 backbone (Fig. 4b). Single framework back-mutations (VH-T71A; VL-Y49S) were introduced to create two variants (Hm-E16.1 and Hm-E16.2) and combined to create a third variant (Hm-E16.3). The resulting humanized VH and VL were combined with human γ1 and κ constant regions, fused to an IgG signal sequence and inserted into expression plasmids. To construct the chimerized antibodies, the mouse VH and VL sequences were combined with the human γ1 and κ constant regions.

Figure 4
Construction and efficacy of humanized E16. A and B. Sequence and alignment of VH and VL of mouse and humanized E16 (Hm-E16.1). Residues VL-49 and VH-71 are underlined and were selected for back-mutation in version 16.2 (VL-Y49L) and 16.3 (VL-Y49L, VH-T71A) ...

We expressed humanized (Hm-E16) and chimerized (Ch-E16) E16 in 293-T cells, and purified them from supernatants by affinity and size exclusion chromatography (data not shown). The affinity was analyzed by surface plasmon resonance using purified antibody in the solid phase. Mouse E16 binds DIII with an affinity of 3.4 nM and a half-life of 3.9 minutes. The affinity of the Ch-E16 and Hm-E16 was similar with KD ranging from 7.1 to 21 nM (Fig. 4c). Hm-E16, Ch-E16, and the parent E16 all had similar PRNT50 values (Fig. 4d).

(b) Effector function of E16

We hypothesized that E16 could also control infection in mice through effector functions including antibody-dependent complement fixation and cytotoxicity. To test this, we generated a Ch-E16 N297Q aglycosyl variant that neutralizes WNV (Fig. 4d) but does not efficiently fix complement or bind Fc γ receptors 28. Mice were administered Ch-E16 or Ch-E16 N297Q at day 2 after WNV infection. Although high doses of Ch-E16 and Ch-E16 N297Q provided virtually complete protection, lower doses of the aglycosyl variant afforded less protection (Fig. 4e). Administration of 4 μg of Ch-E16 resulted in 84% survival whereas 4 μg of Ch-E16 N297Q provided only 31% protection.

To test which effector function enhanced the activity of E16, we performed studies with 8 week-old C1q, C4, or Fc γ R I and III-deficient C57BL/6 mice (Fig. 5). These mice all show increased susceptibility to lethal WNV infection compared to wild type controls. In C1q or C4-deficient mice, which cannot activate complement by the antibody-dependent classical pathway, E16 had a similar potency compared to wild type mice. In contrast, in Fc γ R-deficient mice, although high doses afforded complete protection, lower doses resulted in higher mortality rates. A dose of 20 μg at day 2, which strongly protected wild type, C1q or C4-deficient mice, did not improve the survival rate of Fc γ R-deficient mice (P = 0.4). Thus, the Fc region enhances the potency of E16 in mice, by virtue of its ability to bind to Fc γ receptors.

Figure 5
Efficacy of mouse E16 in complement and Fc γ R-deficient mice. Dose-response analysis at day 2 after WNV infection. 8 week-old (A) Fc γ R I and III-deficient, (B) C1q-deficient, or (C) C4-deficient C57BL/6J mice were inoculated with 10 ...

(c) Therapeutic activity of Hm-E16

To confirm the efficacy of humanized E16, wild type mice were administered three different versions of purified Hm-E16 at day 2 after infection. Although Hm-E16 variants protected mice against lethal infection (Fig. 4f), at a 4 μg dose, the variant (16.3) that demonstrated the highest affinity for DIII (Fig. 4c) was more protective (67% versus 46% survival; mean survival time of 14 ± 1 d versus 11 ± 2 d, P = 0.04).


We generated a panel of 46 mAbs against WNV E protein, and applied a novel high-throughput epitope mapping strategy to identify a dominant epitope that was recognized by the majority of neutralizing mAbs in DIII. This epitope was also detected by convalescent antibodies from human patients who had recovered from WNV infection without clinical consequence. Three neutralizing mAbs protected against WNV mortality in a post-exposure therapy model. One of these, E16, was humanized and confirmed as therapeutically effective in mice.

Previous studies have mapped amino acid contact residues of neutralizing mAbs by sequencing in vitro neutralization escape variants, through site-specific substitution of specific charged or polar residues, and by performing binding assays with overlapping peptide libraries. We used error prone PCR mutagenesis and yeast surface expression to identify contact residues in a high-throughput manner. Although this technique has been used previously 29, this is the first high-throughput epitope mapping application. By having a large panel of DIII mAbs and selecting only variants that abolished or markedly reduced binding of a few mAbs, we minimized the possibility that mutations altered protein folding. We have recently confirmed the recognition sites on DIII for E16 and the validity of the yeast display strategy by solving the crystal structure of the E16 Fab-DIII complex (G. Nybakken, T. Oliphant, M. Diamond, and D. Fremont, manuscript submitted).

Ten of twelve neutralizing mAbs localized to the distal-lateral surface of DIII, results that are consistent with prior studies that mapped three neutralizing mAbs against WNV using in vitro escape variants 15,30. Our weakly and non-neutralizing mAbs did not recognize this epitope but localized to distinct regions. E16 recognized the dominant epitope and neutralized all strains that were tested. Sequence analysis of 124 WNV strains in public databases revealed almost complete (98.4 to 100%) conservation of the contact residues S306, K307, T330, and T332. Only two clinically attenuated lineage II isolates had mutations at these residues. Because of the structural homology among flaviviruses 7,8,16, the analogous amino acids that map to the distal lateral surface of DIII can be readily identified. Although this region is highly variable among flaviviruses, most mutations that abolish binding of virus-specific neutralizing antibodies map here 15,18,30,31, suggesting the existence of an analogous dominant neutralizing epitope for other flaviviruses. We speculate that successful vaccines against WNV or other flaviviruses should induce potent humoral responses against this neutralizing epitope.

Although passive administration of immune human γ–globulin after WNV infection improved survival in mice 4,5, it may be limited by its low-titer neutralizing activity, variability, and risk of transmission of infectious agents. Only two prior studies have demonstrated post-exposure therapy of neutralizing mAbs with flaviviruses: 6B5A-2 reduced mortality 3 to 4 days after Saint Louis encephalitis virus infection 6; and 503 reduced Japanese encephalitis virus mortality 5 days after infection 32. Here, we show that three different neutralizing mAbs improved survival even when administered 4 and 5 days after WNV infection. Moreover, therapy with E16 at day five completely cleared WNV from the brain at day 9 in 68% of mice. Thus, inhibitory WNV mAbs improve clinical and virologic outcome even after CNS viral spread, results that agree with studies showing that antibody can mediate viral clearance from infected neurons 33,34.

Our experiments are consistent with a model in which the therapeutic efficacy of mAbs is determined by properties in addition to neutralization: (a) The mAb (E24) with the strongest neutralizing activity in vitro did not have the greatest efficacy in vivo; (b) An aglycosyl version of E16 that lacked the ability to fix complement or bind to Fc γ receptors had equivalent neutralizing but reduced therapeutic activity; (c) E16 was less potent in mice that lacked Fc γ receptors.

E16 was humanized as a possible therapeutic for humans. Hm-E16 bound DIII with similar affinity and demonstrated efficacy as post-exposure therapy. Moreover, it may be possible to improve Hm-E16 by introducing mutations that enhance affinity, creating forms of E16 that more readily cross the blood-brain barrier, and combining mAbs that neutralize WNV infection through independent mechanisms. Our results are the first successful demonstration of a humanized mAb as post-exposure therapy against a viral disease, and suggest that antibody-based therapeutics may have more broad utility than previously appreciated, especially in the treatment of CNS infections in which an effective antibody response is important for limiting virus dissemination and injury to neurons.


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).

Mouse experiments

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+ and Mg2+ 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 4547 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.

Statistical Analysis

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.

Supplementary Material

Supp figs

Supp tables


The authors thank A. Pekosz, K. Blight, D. Leib, L. Morrison, R. Klein, P. Olivo, and T. Pierson and their laboratories for experimental advice. The authors thank H. Virgin and D. Goldberg for critical reading of the manuscript, G. Stahl and M. Carroll for complement-deficient mice, and M. Busch and L. Tobler for the human plasma samples. The work was supported by grants from NIH (U01 AI061373 to M.S.D. and U54 AI057160 to the Midwest Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research), the Pediatric Dengue Vaccine Initiative, the Edward Mallinckrodt Jr. Foundation and a New Scholar Award in Global Infectious Diseases from the Ellison Foundation. Chris Doane was supported in part by a fellowship funded by an Undergraduate Biological Sciences Education Program grant from the Howard Hughes Medical Institute to Washington University.


1. Granwehr BP, et al. West Nile virus: where are we now? Lancet Infect Dis. 2004;4:547–56. [PubMed]
2. Diamond MS, Shrestha B, Mehlhop E, Sitati E, Engle M. Innate and adaptive immune responses determine protection against disseminated infection by West Nile Encephalitis virus. Viral Immunology. 2003;16:259–278. [PubMed]
3. Wang T, Fikrig E. Immunity to West Nile virus. Curr Opin Immunol. 2004;16:519–23. [PubMed]
4. Ben-Nathan D, et al. Prophylactic and therapeutic efficacy of human intravenous immunoglobulin in treating west nile virus infection in mice. J Infect Dis. 2003;188:5–12. [PubMed]
5. Engle M, Diamond MS. Antibody prophylaxis and therapy against West Nile Virus infection in wild type and immunodeficient mice. J Virol. 2003;77:12941–12949. [PMC free article] [PubMed]
6. Roehrig JT, Staudinger LA, Hunt AR, Mathews JH, Blair CD. Antibody prophylaxis and therapy for flaviviral encephalitis infections. Ann NY Acad Sci. 2001:286–297. [PubMed]
7. Modis Y, Ogata S, Clements D, Harrison SC. A ligand-binding pocket in the dengue virus envelope glycoprotein. Proc Natl Acad Sci U S A. 2003;100:6986–91. [PubMed]
8. Rey FA, Heinz FX, Mandl C, Kunz C, Harrison SC. The envelope glycoprotein from tick-borne encephalitis virus at 2 Angstrom resolution. Nature. 1995;375:291–8. [PubMed]
9. Rey FA. Dengue virus envelope glycoprotein structure: new insight into its interactions during viral entry. Proc Natl Acad Sci U S A. 2003;100:6899–901. [PubMed]
10. Modis Y, Ogata S, Clements D, Harrison SC. Structure of the dengue virus envelope protein after membrane fusion. Nature. 2004;427:313–9. [PubMed]
11. Zhang, W. et al Visualization of membrane protein domains by cryo-electron microscopy of dengue virus. Nat Struct Biol (2003). [PubMed]
12. Mukhopadhyay S, Kim BS, Chipman PR, Rossmann MG, Kuhn RJ. Structure of West Nile virus. Science. 2003;302:248. [PubMed]
13. Bhardwaj S, Holbrook M, Shope RE, Barrett AD, Watowich SJ. Biophysical characterization and vector-specific antagonist activity of domain III of the tick-borne flavivirus envelope protein. J Virol. 2001;75:4002–7. [PMC free article] [PubMed]
14. Chu JJ, et al. Inhibition of West Nile virus entry by using a recombinant domain III from the envelope glycoprotein. J Gen Virol. 2005;86:405–12. [PubMed]
15. Beasley DW, Barrett AD. Identification of neutralizing epitopes within structural domain III of the West Nile virus envelope protein. J Virol. 2002;76:13097–13100. [PMC free article] [PubMed]
16. Volk, D.E. et al Solution structure and antibody binding studies of the envelope protein domain III from the New York strain of West Nile virus. J Biol Chem (2004). [PubMed]
17. Beasley DW, Aaskov JG. Epitopes on the dengue 1 virus envelope protein recognized by neutralizing IgM monoclonal antibodies. Virology. 2001;279:447–58. [PubMed]
18. Crill WD, Roehrig JT. Monoclonal antibodies that bind to domain III of dengue virus E glycoprotein are the most efficient blockers of virus adsorption to Vero cells. J Virol. 2001;75:7769–73. [PMC free article] [PubMed]
19. Roehrig JT, Bolin RA, Kelly RG. Monoclonal antibody mapping of the envelope glycoprotein of the dengue 2 virus, Jamaica. Virology. 1998;246:317–28. [PubMed]
20. Cecilia D, Gould EA. Nucleotide changes responsible for loss of neuroinvasiveness in Japanese encephalitis virus neutralization-resistant mutants. Virology. 1991;181:70–7. [PubMed]
21. Wu SC, Lian WC, Hsu LC, Liau MY. Japanese encephalitis virus antigenic variants with characteristic differences in neutralization resistance and mouse virulence. Virus Res. 1997;51:173–81. [PubMed]
22. Lin B, Parrish CR, Murray JM, Wright PJ. Localization of a neutralizing epitope on the envelope protein of dengue virus type 2. Virology. 1994;202:885–90. [PubMed]
23. Diamond MS, Shrestha B, Marri A, Mahan D, Engle M. B cells and antibody play critical roles in the immediate defense of disseminated infection by West Nile encephalitis virus. J Virol. 2003;77:2578–2586. [PMC free article] [PubMed]
24. Smithburn KC, Hughes TP, Burke AW, Paul JH. A neurotropic virus isolated from the blood of a native of Uganda. Am J Trop Med Hyg. 1940;20:471–492.
25. Boder ET, Wittrup KD. Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol. 1997;15:553–7. [PubMed]
26. LoBuglio AF, et al. Mouse/human chimeric monoclonal antibody in man: kinetics and immune response. Proc Natl Acad Sci U S A. 1989;86:4220–4. [PubMed]
27. Meredith RF, et al. Pharmacokinetics, immune response, and biodistribution of iodine-131-labeled chimeric mouse/human IgG1,k 17-1A monoclonal antibody. J Nucl Med. 1991;32:1162–8. [PubMed]
28. Tao MH, Morrison SL. Studies of aglycosylated chimeric mouse-human IgG. Role of carbohydrate in the structure and effector functions mediated by the human IgG constant region. J Immunol. 1989;143:2595–601. [PubMed]
29. Shusta EV, Holler PD, Kieke MC, Kranz DM, Wittrup KD. Directed evolution of a stable scaffold for T-cell receptor engineering. Nat Biotechnol. 2000;18:754–9. [PubMed]
30. Chambers TJ, Halevy M, Nestorowicz A, Rice CM, Lustig S. West Nile virus envelope proteins: nucleotide sequence analysis of strains differing in mouse neuroinvasiveness. J Gen Virol. 1998;79 ( Pt 10):2375–80. [PubMed]
31. Wu, K.P. et al Structural basis of a Flavivirus recognized by its neutralizing antibody: Solution structure of the domain III of the Japanese Encephalitis virus envelope protein. J Biol Chem (2003). [PubMed]
32. Kimura-Kuroda J, Yasui K. Protection of mice against Japanese encephalitis virus by passive administration with monoclonal antibodies. J Immunol. 1988;141:3606–3610. [PubMed]
33. Virgin HWt, Bassel-Duby R, Fields BN, Tyler KL. Antibody protects against lethal infection with the neurally spreading reovirus type 3 (Dearing) J Virol. 1988;62:4594–604. [PMC free article] [PubMed]
34. Levine B, et al. Antibody-mediated clearance of alphavirus infection from neurons. Science. 1991;254:856–60. [PubMed]
35. Diamond M, et al. Modulation of dengue virus infection in human cells by alpha, beta, and gamma interferons. . J. Virol. 2000;74:4957–4966. [PMC free article] [PubMed]
36. Ebel GD, Carricaburu J, Young D, Bernard KA, Kramer LD. Genetic and phenotypic variation of West Nile virus in New York, 2002–2003. Am J Trop Med Hyg. 2004;71:493–500. [PubMed]
37. International Catalogue of Arboviruses, (American Society of Tropical Medicine and Hygiene, San Antonio, 1985).
38. Delenda C, Staropoli I, Frenkiel MP, Cabanie L, Deubel V. Analysis of C-terminally truncated dengue 2 and dengue 3 virus envelope glycoproteins: processing in insect cells and immunogenic properties in mice. J Gen Virol. 1994;75:1569–78. [PubMed]
39. Lanciotti RS, et al. Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States. Science. 1999;286:2333–7. [PubMed]
40. Harlow, E. & Lane, D. Antibodies, A laboratory manual, 714 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988).
41. Boder ET, Wittrup KD. Optimal screening of surface-displayed polypeptide libraries. Biotechnol Prog. 1998;14:55–62. [PubMed]
42. Kabat, E.A., Wu, T.T., Perry, H., Gottesman, K. & Foeller, C. Sequences of proteins of immunological interest, 3242 (NIH, 1991).
43. Matsuda F, et al. The complete nucleotide sequence of the human immunoglobulin heavy chain variable region locus. J Exp Med. 1998;188:2151–62. [PMC free article] [PubMed]
44. Ravetch JV, Siebenlist U, Korsmeyer S, Waldmann T, Leder P. Structure of the human immunoglobulin mu locus: characterization of embryonic and rearranged J and D genes. Cell. 1981;27:583–91. [PubMed]
45. Klobeck HG, Meindl A, Combriato G, Solomon A, Zachau HG. Human immunoglobulin kappa light chain genes of subgroups II and III. Nucleic Acids Res. 1985;13:6499–513. [PMC free article] [PubMed]
46. Klobeck HG, et al. Subgroup IV of human immunoglobulin K light chains is encoded by a single germline gene. Nucleic Acids Res. 1985;13:6515–29. [PMC free article] [PubMed]
47. Hieter PA, Maizel JV, Jr, Leder P. Evolution of human immunoglobulin kappa J region genes. J Biol Chem. 1982;257:1516–22. [PubMed]