Human Monoclonal Fab Antibodies Against West Nile Virus and its
Journal of Antivirals & Antiretrovirals

Journal of Antivirals & Antiretrovirals
Open Access

ISSN: 1948-5964

Research Article - (2009) Volume 1, Issue 1

Human Monoclonal Fab Antibodies Against West Nile Virus and its Neutralizing Activity Analyzed in vitro and in vivo

Tao Duan1, Monique Ferguson2, Lintian Yuan3, Fangling Xu1 and Guangyu Li2*
1Department of Pathology and Center for Biodefense and Emerging Infectious Diseases, USA, E-mail:
2Department of Internal Medicine, Division of Infectious Diseases, University of Texas Medical Branch, 301 University Blvd, Galveston, TX, USA 77555-0435, USA, E-mail:
3Department of Pediatric Dentistry, College of Stomatology, Fourth Military Medical University, Xi�??an, China 710032, China, E-mail:
*Corresponding Author: Dr. Guangyu Li, M.D. & Ph.D., Department of Internal Medicine, Division of Infectious Diseases, University of Texas Medical Branch, 301 University Boulevard,Galveston, TX 77555-0435, China, Tel: (409) 747-0275, Fax: (409) 772- 6527 Email:


The disease progression with West Nile virus (WNV) infection in humans leads to meningitis or encephalitis and may cause death, particularly among elderly and immunocompromised individuals. Passive immunity using immunoglobulins has shown efficacy in treating some patients with WNV infection, which makes the development of human anti-WNV antibodies significant. The goal of this study was to construct a Fab-specific phage display library against WNV, and to identify and select clones with neutralizing activities. Total RNA was extracted from peripheral blood lymphocytes (PBLs) of two immunized individuals, and RT-PCR was used to amplify the Fab fragments containing the heavy (VH) and light (VL) chains. The amplified genes were sequentially cloned into the recombinant antibody expression vector pComb3-H, and the Fab-specific phage display library was packaged with helper phage VCS-M13. Five rounds of panning were carried out with WNV E protein domain III, and then binding antibodies were selected by ELISA.


Keywords: Tao Duan, Monique Ferguson, Lintian Yuan, Fangling Xu, Guangyu Li


West Nile virus (WNV) is a single-stranded, positive-polarity RNA flavivirus that is related to viruses causing dengue fever, yellow fever, St. Louis, tick-borne and Japanese encephalitis. Human infections with WNV develop a febrile illness that can progress to meningitis or encephalitis and may lead to death, particularly among those elderly and immunocompromised (Granwehr et al., 2004; Marfin and Gubler, 2001). The clinical manifestations of WNV infection are well defined, but the mechanism of pathogenesis has not been elucidated completely. Previous studies have proven that WNV could infect and induce cytopathogenicity in various cell cultures of human, primate, rodent and insect origin. Both necrosis and apoptosis in WNV-infected cells and tissues were observed in patients, as well as in experimental animal models of fatal WNV infections (Xiao et al., 2001).

Currently, treatment is supportive and no approved vaccine exists for clinical use. The innate and adaptive immune responses can prevent WNV dissemination within the central nervous system (CNS) (Diamond et al., 2003), and the antiviral antibody may work directly in the CNS by preventing replication and spread in neurons (Agrawal and Petersen, 2003). Recently, various groups showed therapeutic efficacy of immune human γ- globulin and humanized monoclonal antibody in mice infected with WNV (Agrawal and Petersen, 2003; Engle and Diamond, 2003; Oliphant et al., 2005; Tesh et al., 2002; Gould et al., 2005). The passive administration of immune γ-globulin or monoclonal antibody improved survival even after virus had spread to the CNS (Engle and Diamond, 2003; Oliphant et al., 2005). These results suggest that a potent neutralizing monoclonal antibody could represent another potential direction to influence disease outcome.

Most neutralizing antibodies against flaviviruses recognize the envelope (E) glycoprotein. Monoclonal antibodies produced against the E protein have been found to protect mice from lethal infection (Oliphant et al., 2005; Gould et al., 2005; Nybakken et al., 2005; Kaufmann et al., 2006; Pereboev et al., 2008). Crystallographic analysis of the soluble ectodomain of flavivirus E proteins has shown that there are three domains. Domain I is an eight-stranded β-barrel which participates in the conformational changes associated with the acidification in the endosome. Domain II contains 12 β-strands and has roles in dimerization, trimerization and fusion (Modis et al., 2003; Rey et al., 1995; Rey, 2003; Modis et al., 2004). Domain III adopts an immunoglobulin- like fold, and contains surfaced exposed loops which putatively play a role in receptor attachment in the mature virion (Mukhopadhyay et al., 2003; Chu et al., 2005; Bhardwaj et al., 2001). Many neutralizing antibodies against flaviviruses recognize Domain III of E protein.

There is an urgent need to develop human antibodies against WNV which could be used for therapeutic purposes. Based on the importance of Domain III of E protein, we aimed to develop human antibodies against this domain.

In this study, we constructed Fab antibody phage display library generated from the PBLs of immunized donors, and obtained human Fab antibodies binding to WNV E protein domain III. We evaluated the neutralizing activities of three antibodies which have high binding activities in vitro and further evaluated the protection efficiency of one antibody in vivo.

Materials and Methods

Cells and Viruses

Vero cells (ATCC CCL-81) were cultured as previously described (Mou et al., 2006). We performed neutralization experiments with the WNV strain Egypt 101 (Samoilova et al., 2003), and the titer of the WNV strain was 107 PFU/ml, and animal experiments with the WNV strain NY385-99 (Melnick et al., 1951).

RNA Preparation and Construction of Expression Vector

Ten milliliters of blood was drawn from two individuals with high neutralizing antibody titer against WNV. Lymphocytes were isolated by centrifugation with a lymphocyte separation medium (Pharmacia). Total RNA was isolated using Trizol reagent (Invitrogen) and was used to synthesize first-strand cDNAs using Superscript III™ first-strand synthesis system for RT-PCR (Invitrogen). DNA of the antibody Fab portion was amplified using specific primers for the antibody heavy- and light-chain genes. The primers were designed according to the previous study (Barbas and Burton, 1994) (data not shown). The VH region of the heavy chains and the light chains were amplified. PCR was performed as as follows: 35 cycles of denaturing at 95°C for 1 min, annealing at 52°C for 1 min, and extending at 72°C for 2 min followed by a final incubation at 72°C for 10 min. The amplified light chains were digested with XbaI and SacI, and purified by electrophoresis in 1.5% agarose gel. The relevant DNA bands were excised from the gels and extracted using a QIAquick gel extraction kit (Qiagen). Purified DNAs were ligated with XbaI / SacI - linearized pComb3-H vector (provided by the Scripps Research Institute). Heavy chain Fd fragments were cut with excess of the restriction enzymesXho I and Spe I and were cloned into Xho I / Spe I – linearized pComb3-H harboring light chains. The DNA pellet was transformed into electrocompetent E.coli XL1-Blue cells (Invitrogen). After transformation, XL1-blue -DNA mix (10, 1, 0.1 μl) was plated to determine the efficiency of transformation. The insertion of target genes was detected by digestion with specific enzymes from the plasmids which were purified from several random XL1-blue monoclones.

Packaging of Phage Libraries

Following electroporation, the library was cultured in 50 ml 2×YT medium with 100 μg/ml ampicillin, 30 μg/ml tetracycline, 100 mM glucose to OD600 value of 0.025. The culture was incubated at 37°C for 2 h. Helper phage VCS-M13 (1012 pfu, Stratagene) was added at a m.o.i. of 8 at OD600 value of 0.1. The culture was continued at 80 rpm for 30 min and at 260 rpm for 30 min. The culture was centrifuged at 4000 rpm and the medium was changed to 40 ml 2×YT with 100 μg/ml ampicillin and 50μg/ml kanamycin. The culture was further incubated for 6 h at 30°C, then centrifuged. Supernatant was added with PEG to 4% and NaCl to 3%. The phage library was precipitated at 4°C overnight before it was centrifuged at 8000 rpm for 30 min. The phage pellet was resuspended in PBS (pH 7.4) and transferred to microcentrifuge tubes at 14000 rpm for 10 min to remove insoluble material. The Fab phage display library was then used for panning experiments.

Panning of anti-WNV Fab Phage Display Library

Several 96-well plates were coated with 200 μl highly purified recombinant WNV envelope protein containing domain III (500ng/well), which was provided by Dr. David Beasley (University of Texas Medical, Galveston, TX), and was incubated at 4°C overnight. After blocking with 3% BSA at 37°C for an hour, 50 μl phage suspensions were added to each well (total of about 107 pfu). The panning procedure is a modification of procedure originally described by Parmley and Smith (Barbas and Lerner, 1991). Following 5 rounds of panning, the percent yield of phage was determined as (No. of phage eluted/No. of phage applied) × 100.

ELISA Analysis of Fab Antibodies

Fab clones were selected and cultured in 2YT medium overnight, then the cultures were induced by 1 mM isopropylbeta- D-thiogaractopyranoside (IPTG). The cells were recovered by centrifugation, and re-suspended in PBS. Fab antibodies were extracted by freezing at -70°C and thawing at 4°C for three times, then were centrifuged at 10,000 rpm and the supernatants were collected. Ninety-six well plates were coated with 100 μl highly purified recombinant WNV envelope protein containing domain III and blocked with 3% BSA as described above. After extensive washing with PBST, the supernatants of Fab antibody were added to the wells (50 μl / well) and incubated at 37°C for 2h. Following 3 washes with PBST, 50 μl of a 1:20,000 dilution of horseradish peroxidase (HRP) conjugated goat anti-human Fab (Sigma, Ronkonkoma, NY) was added and incubated at 37°C for 1 h. Finally, 50 μl of TMB (Sigma, Ronkonkoma, NY) was added and color development was monitored at 490nm. The A490 values of positive clones were higher than that of negative clones at least two-fold.

Sequence Analysis of WNV-specific Fab Clones

Nucleotide sequences of the heavy and light chain variable regions of the three positive clones (Granwehr et al., 2004; Modis et al., 2003; Haard et al., 1999) were determined by the University of Texas Medical Branch Protein Chemistry Laboratory. DNA and deduced amino acid sequences were analyzed by using Pubmed Igblast Software ( Further analysis of amino acid sequences of VH and VL of WNV-specific human Fab antibodies was performed according to (Kim et al., 2004).

Western Blotting Analysis of WNV-specific Fab Antibodies

Maltose binding protein (MBP) WNV domain III was heated to 100°C for 10 min in equal volume of 2x SDS gel-loading buffer, and loaded up to 20 μl of each of the samples into the bottom of the wells for electrophoresis (100V for about 4 h until the bromophenol blue reached the bottom of the resolving gel). Electrophoresis was performed in duplicates. One gel was fixed with glacial acetic acid: methanol: water (10:20:70) for 10 min and washed in deionized water, then stained with Coomassie Brilliant Blue, the other was used for Western blot analysis. The proteins were transferred to nitrocellulose membrane, and nonspecific binding sites were blocked with nonfat dried milk by incubating the membrane for 2 h at room temperature with gentle agitation on a platform shaker. The membrane was washed with TBST for 3 times (10 min each). Then the membrane paper was cut into five strips according to the lanes, and respectively incubated with 1:2 dilution of antibodies Fab 1, Fab 13, Fab 25 and 1:200 dilution of positive serum for 2 h at room temperature with gentle agitation on a platform shaker, and after washing as above, membranes were incubated with 1:20,000 dilution of HRP-conjugated goat anti-human Fab for 1 h, and were detected by ECL Western blotting detection reagent (Amersham), The membranes were exposed to X-ray film for 30 sec, and the film was developed immediately.

Indirect Fluorescent Antibody (IFA) Assay to Detect WNV in Vero 76 Cells

Sterilized round cover slips were placed into 6-well plates, and Vero 76 cells (5× 105 cells/well) were seeded into the plates and cultured for 20 h. Cells were inoculated with 100 μl of virus suspension in test medium containing equal volumes of WNV (approximately 100 plaque forming units [pfu] ), and incubated at 37°C for 1 h. The inocula were aspirated and fresh MEM medium containing 2% FBS were added. Sixty hours later, the cover slips were carefully removed and examined by IFA assay. Fab 1, 13, and 25, diluted by 1: 100 in PBS, human convalescent WNV serum, diluted by 1:200 in PBS, and human pooled immune globulin (without WNV antibody), diluted by 1:200 in PBS, were added to each slides and incubated at 37°C for 1 h. These were washed with PBST and incubated at 37°C for 50 min with 50 μl of fluorescein isothiocyanate (FITC)-conjugated goat anti-human Fab (Sigma, St. Louis, MO), diluted 1:60 in PBS and containing 1% Evan’s blue dye. Evidence of specific fluorescence was monitored by fluorescence microscopy (microscopic field 20X), using an Olympus BX51 microscope.

Plaque Reduction Neutralization Tests (PRNT) for Analysis of Neutralizing Activity of Fab Antibodies

This assay was performed in triplicates. Briefly, after removal of the cell growth medium, confluent 24 h Vero 76 cell monolayer in 24-well plates (Becton Dickinson labware, NJ USA) were inoculated with 100 μl of the respective virus suspension in test medium containing equal volumes of WNV (approximately 100 pfu and serial two-fold dilutions of the test human recombinant antibodies, which had been mixed and inoculated at 37°C for 1 h. After adsorption at 37°C and 5% CO2 for 1 h, the inocula were aspirated and each well was overlayed with 1 ml mixture of MEM medium containing 2% FBS and 1% carboxymethyl-cellulose(Sigma).Heat-inactivated anti-WNV human positive/negative serum and recombinant plasmid were used as positive, negative and mock controls at the same time. Three untreated virus controls and one uninfected cell control were included in all assays. Each compound concentration was tested in duplicate. The tests were incubated at 37°C and 5% CO2 for 72 h until plaques appeared and fixed by 10% formalin for 48 h (changing once after 24 h) and then stained with a solution of 0.5% crystal violet in PBS. Plaques were counted over a light box after removal of the crystal violet. Neutralizing antibody activity was considered as the concentration of the antibody dilution with an 80% reduction in the number of plaques (PRNT80), as compared to the virus control and other a series of controls.

Mice Passive Immunization and Viral Challenge

Eight groups of 32 female C57BL/6 mice (Jackson Laboratories) between 4 and 6 weeks age were used in this experiment. Mice were infected with lethal dose WNV strain NY385-99 (103 PFU) intraperitoneally (i.p.) on day 1, and administered with the indicated doses of serum (positive control, negative control) or Fab 1 at times ranging from 1 day prior to 1 day post infection (Table 1). Survivals were recorded daily until no further deaths occurred for at least 21 days after infection.


Cloning of Anti-WNV Fab Genes

A mixture of PCR-amplified k/λ-chain products that had been digested by the appropriate restriction enzymes were ligated to the pComb3-H vector and introduced into E. coli XL1-Blue by electroporation. Titration ampicillin-resistant clones indicated that the light chain library contained 5×106 independent clones. PCR-amplified heavy chain products were ligated to DNAs extracted from the light chain library to generate a phage display Fab library with 2×107 clones. To examine the authenticity of the library, 30 clones were picked at random and analyzed. Light chain and heavy chain insert efficiency was approximately 53.3% and 33.3%, respectively.

ELISA Analysis of Anti-WNV Fab Antibodies

The library was panned to select clones which have binding activities to WNV domain III antigen. After 5 rounds of panning, phagemid DNAs obtained were introduced into E. coli XL1-Blue to develop Fab antibodies, and each clone was tested by ELISA; 8 clones (Fab 1, 6, 13, 16, 22, 23, 24, 25) which have Fab antibody proteins showed binding activities to WNV domain III protein in ELISA; three (Fab 1, 13, 25) had higher affinity than the others (Figure 1).


Figure 1: ELISA analysis of Fab antibodies against WNV E protein domain III.

Sequence Analysis of WNV-specific Fab Clones

Sequence analysis of the heavy and light chain variable regions of Fab 1, 13, 25 clones showed that their heavy chain variable region (VH) sequences which include complementarity determining regions (CDRS) that directly interact with the epitope of the antigen were significantly different from each other. They originated from different germline VH segments and also had somatic hypermutations. They belonged to VH1 (Fab 25) and VH3 (Fab 1, 13) gene family, and the light chain variable region (VL) sequences were also highly different to each other and originated from human Vk1 gene family. The results of the sequence analyses are shown in Figure 2.


Figure 2: Amino acid sequences of VL and VH of anti-WNV-E specific human Fabs Amino acid sequences were derived from the DNA sequences of the Fabs. Shown are the framework regions 1 to 4 (FWR1 to FWR4) and complementarity-determining regions 1 to 3 (CDR1 to CDR3) for VH and VL.

Western Blotting Analysis of Specificity and Affinity of Fab Antibodies

The three positive clones (Fab 1, 13, 25) with higher activities in ELISA (Figure 1) were further identified by Western blotting and IFA assay for their specificity and affinity. The results showed each antibody binding specifically to the WNV protein domain III, and WNV proteins. Fab 1 showed the strongest activity compared to Fab 13, and Fab 25 (Figure 3 A and B).


Figure 3: A. Western blotting analysis of Fab antibodies against WNV domain III, B. IFA assay to detect WNV in Vero cells. M: protein standards; P: positive control, human convalescent WNV serum (T-35582); N: human pooled immune globulin (without WNV antibody).

Neutralizing Activity of Fab Antibodies in Vitro

The three clones (Fab 1, 13, 25) showing specificity and affinity to WNV protein domain III were further analyzed by performing neutralization assay. The experiments were repeated three times and the results of each time were consistent. The neutralizing activity associated with crude Fab antibodies was estimated by observing cytopathic effect (CPE) of Vero cells along with a series of controls. Fab 1 antibody exhibited significant neutralizing activity and blocked 100 pfu WNV infection at a concentration of 80 ug/ml (Figure 4), however, Fab 13 and Fab 25 antibodies showed weak neutralizing activity, and modestly blocked 100 pfu WNV infection at a concentration of 320 μg/ml and 80 μg/ml, respectively (Figure 4). In the PRNT, Fab 1 (PRNT80 = 80 ug/ml), Fab 13 (PRNT80 = 320 ug/ml), and Fab 25 (PRNT80 = 160 ug/ml) inhibited infection slightly than positive control (anti-WNV serum). The results demonstrated that the Fab antibodies could neutralize the Egypt 101 strain of WNV and the neutralizing activities of the antibodies were correlated with their affinities to WNV E protein.


Figure 4: PRNT for virus neutralization by anti-WNV Fab antibodies. The “virus control” represents the result obtained by infection of virus only without addition of antibody. The “Negative Serum Control”, “Mock Control”, and “Positive Serum Control” represent the results obtained by the mixtures of virus with no anti-WNV antibody serum, supernatant of lytic XL1-blue, and anti-WNV serum, respectively. The final concentration of antibody in the virus-antibody mixture is indicated.

Protection of Fab Antibody in Vivo

To further examine the ability of the antibodies to neutralize WNV, Fab 1 was tested in vivo in a prophylactic viral infection model system using C57/BL/6 mice as described previously (Xiao et al., 2001). All mice were infected on day 1, and post infection, the animals were observed for 21 days. Deaths occurred between days 8-14, and mortality rates for each group are shown in Table 1, indicating no protection for Fab 1 in mice

Groups Virus only Serum PC Serum NC Fab1 Mortality Rate
A *       4/4
B   *     0/4
C   *     0/4
D   *     0/4
E     *   3/4
F       * 3/4
G       * 3/4
H       * 4/4

Note: Thirty-two female C57/BL/6 mice (4-6 weeks of age) were inoculated with 103 PFU of West Nile virus strain NY385-99, via IP. There were 4 mice per group, as shown in the table above:
A = Virus control (virus but no antibody)
B, C, and D = Received human convalescent WNV serum (T-35582), 200 uL of a 1:2 dilution given IP, one day prior to infection (B), day of infection (C) and 24 hours after infection (D).
E = Human pooled immune globulin (without WNV antibody); 200 uL of 1:2 dilution given IP given 24 hours prior to infection.
F, G and H = Received 200 uL of Fab 1, given IP one day prior to infection (F), day of infection (G) and 24 hours after infection (H).
All mice were infected on day 1. The animals were observed for 21 days post infection. Deaths occurred between days 8-14.

Table 1:Animal experimental design and protection results.


Humoral immune response plays an important role in the control of flavivirus infection and disease. Therapeutic efficacy of immune human γ-globulin and humanized monoclonal antibody in mice infected with WNV were demonstrated by several investigators (Agrawal and Petersen, 2003; Engle and Diamond, 2003; Oliphant et al., 2005; Tesh et al., 2002; Gould et al., 2005). Among them, gene-based delivery of recombinant antibody genes seems to be a promising therapeutic strategy which has the advantages including sustained antibody levels, better safety profile and lower production costs (Kaufmann et al., 2006). Phage display system is powerful tool to generate human genetic antibodies (Haard et al., 1999). Many human genetic antibodies have already been developed with this system, though the mechanism of immune repertories generated in response to acute WNV infection or any flavivirus infection has not been well characterized in humans or primates. Antibodies against Dengue virus has been achieved from antibody phage display repertoires from Dengue virus-infected chimpanzees (Men et al., 2004). The use of partially and completely human antibodies has elicited no or minimal immune response when administered to patients (Holliger and Hoogenboom, 1998; Holliger and Hudson, 2005). Due to the absence of a WNV vaccine for humans, passive immunization represents an important alternative strategy to prevent and treat WNV infection.

In this study, we designed and constructed a phage antibody library specifically to Fab. We used a small volume of peripheral blood (20 ml) from two healthy donors with high WNV antibody titers as source to construct our Fab library. We used total RNA to synthesize the cDNA in the maximum extent. We obtained a phage library with 7×107 clones, which allows the rapid isolation and affinity analysis of antigen-specific human antibody fragments. Three neutralizing Fabs antibodies against WNV envelop protein domain III were developed from our library. These antibodies proved useful for generating Fab antibodies against WNV by plaque reduction neutralization test.

Fab is a construct in which the heavy chain and light chain are joined by a flexible polypeptide linker preventing dissociation. Antibody Fab fragments comprise both VH and VL domains and usually retain the specific, monovalent, antigen-binding affinity of the parent IgG, while showing improved pharmacokinetics for tissue penetration. Many of these products are currently in preclinical studies and clinical trials which supports our strategy in constructing Fab antibody phage display libraries, and selecting and identifying the antibodies against WNV.

The affinities of the selected antibody fragments are dependent on the antigen used for selection. Hoogenboom and colleagues reported an affinity varying between 2.7 and 38 nM for the selected Fab fragments specific for the gonadotropin (Haard et al., 1999), whereas Sheets and colleagues reported the affinity of scFv antibodies to the extracellular domain of human ErbB- 2 varied between 0.22 and 4.03 nM (Sheets et al., 1998). This shows that it is very important to select appropriate antigens for the panning. Therefore, in this study, we used three different recombinant WNV envelope proteins for the panning the highaffinity Fab antibodies, and identified a panel of eight Fab antibodies that bound to the recombinant WNV envelope protein. Among those eight Fab antibodies, three of them were highaffinity antibodies to WNV. The sequences of the antibodies we obtained were blasted in Genebank, and the results showed that the sequences were unique and not previously reported. The heavy chain belong to the IgG1 subclass VH1 and VH3, the light chains belong to k isotype. PRNT showed 80 μg/ml Fab 1 antibody can protect Vero cells from 100 pfu of WNV infection, which demonstrated neutralizing activity.

Passive administration of immune human γ-globulin after WNV infection improved survival in mice (Oliphant et al., 2005; Ben-Nathan et al., 2003; Engle and Diamond, 2003). In contrast our Fab antibody failed to protect mice from death partly because the neutralization potential of an antibody is determined by the strength of binding and abundance of its epitope on the virion (Burton et al., 2001; Oliphant et al., 2007). It may be limited by its own low–titer neutralizing activity, variability, and infectability. The therapeutic efficacy of mAbs is determined by properties in addition to neutralization. One research group found that the mAb with strongest neutralizing activity in vitro did not have the greatest efficacy in vivo, and Fab antibody was less potent in mice that lacked Fc γ receptors (Gould et al., 2005; Sheets et al., 1998); our in vivo data is consistent with the reported study of other research groups (Throsby et al., 2006). Although we found a neutralization potency in vitro, there was no association between potent in vivo activity and in vitro protection. Our experiments suggest that the highly neutralizing antibody has little significant role in primary infection and that the antibody for humans may be skewed toward the induction of cross-reactive and less-neutralization in animal studies. (Roehrig et al., 2001).

Potential conflicts of interest: All authors report no conflicts.

Financial support: National Institutes of Health (NO1 AI- 25489).


We thank Dr. Peter Mason for scientific guidance, Dr. Tesh for providing WNV strains, Dr. David Beasley for presenting WNV envelop protein domain III, and the Scripps Research Institute for kindly providing expression vector pComb3-H.

This work was supported by grants from National Institutes of Health (NO1 AI-25489).


  1. Agrawal AG, Petersen LR (2003) Human immunoglobulin as a treatment for West Nile virus infection. J Infect Dis 188: 1-4.
  2. Barbas III CF, Burton DR (1994) Monoclonal antibodies from combinatorial libraries. Cold Spring Harbor Laboratory Cousre P: 31-35.
  3. Barbas III CF, Lerner RA (1991) Combinatorial immunoglobulin libraries on the surface of phage (phabs): rapid selection of antigen-specific Fabs. Methods 2: 119-124.
  4. Ben-Nathan D, Lustig S, Tam G, Robinzon S, Segal S, et al. (2003) Prophylactic and therapeutic efficacy of human intravenous immunoglobulin in treating west nile virus infection in mice. J Infect Dis 188: 5-12.
  5. Bhardwaj S, Holbrook M, Shope RE, Barrett AD, Watowich SJ (2001) Biophysical characterization and vector-specific antagonist activity of domain III of the tick-borne flavivirus envelope protein. J Virol 75: 4002-4007.
  6. Burton DR, Saphire EO, Parren PW (2001) A model for neutralization of viruses based on antibody coating of the virion surface. Curr Top Microbiol Immunol 260: 109-143.
  7. Chu JJ, Pajamanonmani R, Li J, Bhuvanakantham R, Lescar J, et al. (2005) Inhibition of West Nile virus entry by using a recombinant domain III from the envelope glycoprotein. J Gen Virol 86: 405-412.
  8. Diamond MS, Shrestha B, Mehlhop E, Sitati E, Engle M (2003) Innate and adaptive immune response determine protection against disseminated infection by West Nile Encephalitis virus. Viral Immunol 16: 259-278.
  9. Engle M, Diamond MS (2003) Antibody prophylaxis and therapy against West Nile Virus infection in wild type and immunodeficient mice. J Virol 77: 12941-12949.
  10. Gould LH, Sui JH, Foellmer H, Oliphant T, Wang T, et al. (2005) Protective and therapeutic capacity of human singlechain Fv-Fc fusion proteins against West Nile virus. J Virol 79: 14606-14613.
  11. Granwehr BP, Lillibridge KM, Higgs S, Mason PW, Aronson JF, et al. (2004) West Nile virus: where are we now. Lancet Infect Dis 4: 547-556.
  12. Haard HJD, Neer NV, Reurs A, Hufton SE, Roovers RC, et al. (1999) A large Non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies. J Biol Chem 274: 18218-18230.
  13. Holliger PH, Hoogenboom HR (1998) Antibodies come back from the brink. Nat Biotechnol 16: 1015-1016.
  14. Holliger PH, Hudson PJ (2005) Engineered antibody fragments and the rise of single domains. Nat Biotechnol 23: 1126-1136.
  15. Kaufmann B, Nybakken GE, Chipman PR, Zhang W, Diamond MS, et al. (2006) West Nile virus in complex with the Fab fragment of a neutralizing monoclonal antibody. Proc Natl Acad Sci 103: 12400-12404.
  16. Kim SJ, Jang MH, Stapleton JT, Yoon SO, Kim KS, et al. (2004) Neutralizing human monoclonal antibodies to hepatitis A virus recovered by phage display. Virol 318: 598-607.
  17. Marfin AA, Gubler DJ (2001) West Nile encephalitis: an emerging disease in the United States. Clin Infect Dis 33: 1713-1719.
  18. Melnick JL, Paul JR, Riordan JT, Barnett VH, Goldblum N, et al. (1951) Isolation from human sera in Egypt of a virus apparently identical to West Nile virus. Proc Soc Exp Biol Med 77: 661-665.
  19. Men R, Yamashiro T, Goncalvez AP, Wernly C, Schofield DJ, et al. (2004) Identification of chimpanzee Fab fragments by repertoire cloning and production of a full-length humanized immunoglobulin G1 antibody that is highly efficient for neutralization of dengue type 4 virus. J Virol 78: 4665- 4674.
  20. Modis Y, Ogata S, Clements D, Harrison SC (2003) A ligandbinding pocket in the dengue virus envelope glycoprotein. Proc Natl Acad Sci 100: 6986-6991.
  21. Modis Y, Ogata S, Clements D, Harrison SC (2004) Structure of the dengue virus envelope protein after membrane fusion. Nature 427: 313-319.
  22. Mou DL, Wang YP, Huang CX, Li GY, Pan L, et al. (2006) Cellular entry of Hantaan virus A9 strain: specific interactions with beta3 integrins and a novel 70kDa protein. Biochem Biophys Res Commun 339: 611-617.
  23. Mukhopadhyay S, Kim BS, Chipman PR, Rossmann MG, Kuhn RJ (2003) Structure of West Nile virus. Science 302: 248.
  24. Nybakken GE, Oliphant T, Johnson S, Burke S, Diamond MS, et al. (2005) Structural basis of West Nile virus neutralization by a therapeutic antibody. Nature 437: 764-769.
  25. Oliphant T, Engle M, Nybakken GE, Doane C, Johnson S, et al. (2005) Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus. Nat Med 11: 522-530.
  26. Oliphant T, Nybakken GE, Austin SK, Xu Q, Bramson J, et al. (2007) Induction of Epitope-Specific Neutralizing Antibodies against West Nile Virus. J Virol 81: 11828-11839.
  27. Pereboev A, Borisevich V, Tsuladze G, Shakhmatov M, Hudman D, et al. (2008) Genetically delivered antibody protects against West Nile virus. Antiviral Res 77: 6-13.
  28. Rey FA (2003) Dengue virus envelope glycoprotein structure: new insight into its interactions during viral entry. Proc Natl Acad Sci 100: 6899-6901.
  29. Rey FA, Heinz FX, Mandl C, Kunz C, Harrison SC (1995) The envelope glycoprotein from tick-borne encephalitis virus at 2 Angstrom resolution. Nature 375: 291-298.
  30. Roehrig JT, Staudinger LA, Hunt AR, Mathews JH, Blair CD (2001) Antibody prophylaxis and therapy for flavivirus encephalitis infections. Ann N Y Acad Sci 951: 286-297.
  31. Tesh RB, Arroyo J, Travassos, da Rosa ARA, Guzman H, et al. (2002) Efficacy of killed virus vaccine, live attenuated chimeric virus vaccine, and passive immunization for prevention of West Nile virus ancephalitis in hamster model. Emerg Infect Dis 8: 1392-1397.
  32. Throsby M, Geuijen C, Goudsmit J, Bakker AQ, Korimbocus J, et al. (2006) Isolation and Characterization of Human Monoclonal Antibodies from Individuals Infected with West Nile Virus. J Virol 80: 6982-6992.
  33. Samoilova TI, Votiakov VI, Titov LP (2003) Virologic and serologic investigations of West Nile virus circulation in Belarus. Cent Eur J Public Health 11: 55-62.
  34. Sheets MD, Amersdorfer P, Finnern R, Sargent P, Lindqvist E, et al. (1998) Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens. Proc Natl Acad Sci 95: 6157-6162.
  35. Xiao SY, Guzman H, Zhang H, Travassos, da Rosa APA, et al. (2001) West Nile virus infection in the golden hamster (Mesocricetus auratus): A model for West Nile encephalitis. Emerg Infect Dis 7: 714-721.
Citation: Tao D, Ferguson M, Yuan L, Xu F, Li G (2009) Human Monoclonal Fab Antibodies Against West Nile Virus and its Neutralizing Activity Analyzed in Vitro and in Vivo. J Antivir Antiretrovir 1: 036-042.

Copyright: © 2009 Tao D, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited