Patent Grant
7276334
This invention relates to the construction and characterization of mouse monoclonal antibodies against western equine encephalitis virus (WEE) expressed from hybridoma cell lines.
Western equine encephalitis virus (WEE) is an enveloped positive-sense, single-stranded RNA virus belonging to the alphavirus genus. The 12 kb genome of WEE encodes for nonstructural (5′ end) and structural (3′ end) proteins. The structural proteins are translated from a subgenomic mRNA (26S mRNA) as a polyprotein that is processed by viral and cellular proteases into E1 (53 kDa), E2 (47 kDa), nucleocapsid [NC] (30 kDa), E3 (10 kDa), and 6K (6 kDa) proteins. The E1 and E2 proteins are glycoproteins present in the lipid envelope. The E3 protein is also a glycoprotein that is most often not a component of the virion, but is required for infectivity in wild-type virus. The NC protein encloses the RNA genome in an icosahedral structure. The 6K protein is virion associated and promotes efficient virus assembly (reviewed in Strauss and Strauss, 1994; Strauss et al., 1995; Johnston and Peters, 1996; Schlesinger and Schlesinger, 1996).
WEE is localized to the Western hemisphere and poses a serious hazard to human health. Virus transmission is by infected mosquitoes, causing disease in humans and horses. Symptoms of WEE infection in humans include encephalitis, convulsions, paralysis, malaise, fever, headaches, nausea, and vomiting. The case fatality rate in humans is 2% to 7%. Currently, there are no known antiviral drugs effective against WEE. Although inactivated WEE vaccine exist for use in limited populations such as laboratory personnel who are at high risk of exposure to the virus, the immunogenicity of the inactivated WEE vaccine is often poor and the immunity is short-lived. Better protection against WEE is required (Johnston and Peters, 1996).
Alphavirus antigenic properties and antibody neutralization have been studied with anti-alphavirus antibodies from mouse immunoglobulins. Murine antibodies capable of neutralizing virus have been generated against E1 and E2 (Mathews and Roehrig, 1982; Boere et al., 1983; Yamamoto et al., 1985; Yamamoto, 1986). Mice were protected from challenge with WEE and Venezuelan equine encephalitis virus (VEE) when injected with antibodies against E1 and E2 in passive immunization studies (Mathews and Roehrig, 1982; Hunt and Roehrig, 1985; Yamamoto, 1986). Anti-E2 monoclonal antibodies were able to protect mice from lethal injections of Semliki Forest virus (SFV) (Boere et al., 1983). Furthermore, neutralizing and non-neutralizing antibodies to E1 and E2 administered to mice, before or after infection with virus, were protected from Sindbis virus (SIN) (Griffin et al., 1997).
Animal antisera and monoclonal antibodies provide important sources of antibody. Although recombinant antibodies have the advantages of being produced quickly, economically, and in large quantities (Wright et al., 1992; Hayden et al., 1997; Verma et al., 1998), recombinant antibodies grown in bacterial systems are often improperly folded and nonglycosylated (Wright et al., 1992; Verma et al., 1998). One may favor the use of monoclonal antibodies over recombinant antibodies for a variety of reasons. Hybridoma technology is able to provide a wide range of monoclonal antibodies that bind to different antigens with high specificity and affinity (Winter and Milstein, 1991; Laurino et al., 1999). Furthermore, monoclonal antibodies can be isolated with high purity (Winter and Milstein, 1991; Laurino et al., 1999). Accordingly, production of monoclonal antibodies directed against WEE is desirable.
Up until recently, only a limited number of monoclonal antibodies against WEE existed and have not been fully characterized. For instance, monoclonal antibodies produced by Hunt and Roehrig (1985) are capable of immunoprecipitating the E1/E2 heterodimer, identifying antigenic determinants on E1, and protecting mice when challenged with WEE. Monoclonal antibodies produced by Yamamoto et al. (1985), showing specificity for E1 and E2 in enzyme-linked immunosorbent assays (ELISA), demonstrate neutralizing activity and are found effective in passive immunization studies (Yamamoto et al., 1985; Yamamoto, 1986). Recently, there have been studies directed to specific recombinant antibodies against WEE. For example, Xu et al. (1999) successfully cloned an anti-WEE scFV. In addition, use of recombinant antibodies to histologically stain the cells expressing WEE antigens was reported in Netolitzky et al. (2000). Accordingly, it is advantageous to produce and characterize a group of monoclonal antibodies for use in detecting and diagnosing WEE effectively. It is also advantageous to study the interactions between monoclonal antibodies with other alphaviruses, such as VEE and SIN.
The present invention is directed to the construction and characterization of a group of mouse monoclonal antibodies against WEE.
An object of the present invention is to produce and identify specific monoclonal antibodies displaying various immunological activities against WEE.
Another object of the present invention is to construct and characterize monoclonal antibodies capable of cross-binding to multiple alphaviruses.
It is another object of the present invention to manufacture recombinant antibodies for hydridoma clones expressing anti-WEE monoclonal antibodies.
It is yet a further object of the present invention to use the identified monoclonal antibodies for immunodetection and immunotherpy.
According to one aspect of the present invention, it provides monoclonal antibodies against WEE expressed from hybridomas.
According to another aspect of the present invention, it provides for the construction of recombinant monoclonal antibodies from hybridoma clones against WEE, consisting of the steps of immunizing mice with antigens prepared from WEE infected cells; fusing and cloning hydridoma cells lines from the immunized mice; and genetic engineering recombinant antibodies from said cultured hybridoma cells.
Materials and Methods
Preparation of Mouse Monoclonal Hybridoma Cell Lines
Mice (BALB/c, Charles River) were immunized with three doses of 20 μg of gradient purified, formalin inactivated antigen prepared from WEE strain B11 infected Vero cells (CCL-81, American Type Culture Collection, Manassa, Va.), as previously described (Xu et al., 1999; Long et al., 2000), and 50 μL TiterMax® (CytRx Corp., Norcross, Ga.) adjuvant in a total volume of 100 μl. The injections were given intraperitoneally at three week intervals. Three weeks after the third injection, the mice were given 10 μg of inactivated WEE antigen intravenously, in a total volume of 50 μl in phosphate-buffered saline (PBS). The fusions were performed on spleen cells 5-7 days later. Fusions, initial screening, and subcloning were performed by the Hybridoma Facility, Southern Alberta Cancer Research Centre, University of Calgary, Calgary, Alberta. Hybridoma cell lines were grown and maintained in RPMI 1640 media supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 1× vitamins solution, antibiotic/antimycotic solution (100 units/ml penicillin G, 100 μg/ml streptomycin, and 25 μg/ml amphotericin B), 100 μM nonessential amino acids, and 1 mM sodium pyruvate. All tissue culture reagents were purchased from Gibco BRL, Gaithersburg, Md. The hybridoma cells were maintained at a density of 0.5-1.0×106 cells/ml and doubled approximately every 24 hr.
Purification of Antibodies
Various hybridoma clones producing anti-WEE antibodies (3F3E9G5, 5C5A1H11, 9B10D4D11G4, 10B5E7E2, 11H9E2C12) were cultured in growth media in T150 flasks. Media supernatants were collected at 24 hr time points and used as starting material for antibody purification. The supernatants were passed over protein G columns (Pierce, Rockford, Ill.), which were subsequently washed with ImmunoPure® binding buffer (Pierce). Bound IgG was eluted with ImmunoPure® elution buffer (Pierce) and six 1 ml fractions were collected. All fractions were neutralized with 100 μl of 1 M Tris-HCl pH 7.5 and monitored by absorbance at 280 nm. All antibodies eluted in fractions 3 and 4. The eluted antibodies were further desalted using Excellulose™ columns (Pierce) equilibrated in PBS pH 7.5. Concentrations of antibodies eluted were determined by absorbance (280 nm) measurements (1 mg/ml IgG=A280/1.44).
WEE Indirect ELISA
The WEE indirect enzyme-linked immunosorbent assay (ELISA) was performed as described by Long et al., 1999. In brief, inactivated antigen from WEE strain B11 infected Vero CCL-81 cells was prepared by previous methods (Xu et al., 1999; Long et al., 2000). Varying concentrations of inactivated WEE antigen or BSA were immobilized onto Nunc Maxisorp™ flat bottomed 96 well plates (Gibco BRL). The wells were blocked, washed, and then incubated with mouse monoclonal antibodies for 1 hr at 37° C. The antibodies were diluted to various concentrations in wash buffer consisting of PBS, 0.05% Tween 20, and 0.1% BSA. The wells were subsequently washed and incubated for 1 hr at 37° C. with secondary antibody, horseradish peroxidase-conjugated goat anti-mouse antibody, at a 1:3,000 dilution in wash buffer. The plates were washed and incubated with a 1:1 solution of 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and hydrogen peroxide (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, Md.). The plates were read at an absorbance of 405 nm.
Western Blotting of WEE
Western blotting was performed as described previously (Rice et al., 1994; Long et al., 2000). In brief, 50 μg formalin inactivated WEE antigen (described above) was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). WEE proteins were separated on a 12% discontinuous polyacrylamide gel, after which proteins were transferred from the gel to Immobilon™-P membranes (0.45 μm pore size, PVDF filter type) (Millipore, Bedford, Mass.). The filters were blocked, washed in PBS containing 0.1% Tween 20 and 0.02% SDS, and then probed with anti-WEE mouse monoclonal antibodies for 1 hr at room temperature. The Mini-Protean® II multi-screen apparatus (Bio-Rad Laboratories, Mississauga, Ontario) was used to analyze multiple antibody samples per immunoblot. The primary antibodies used were different dilutions of culture supernatants or different concentrations of purified protein in wash buffer. These blots were washed and incubated with a 1:3,000 dilution of horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (H+L) (Caltag Laboratories, Burlingame, Calif.) for 1 hr at room temperature. Proteins were detected using the enhanced chemiluminescence (ECL) method (Amersham Pharmacia Biotech, Baie D'Urfé, Québec).
Isotyping Antibodies
Isotyping of the antibodies was performed using the monoclonal antibody-based mouse immunoglobulin isotyping kit (Pharmingen, Mississauga, Ontario). Specific rat anti-mouse antibodies (IgG1, IgG2a, IgG2b, IgG3, IgM, IgA, Igκ, and Igλ) were coated onto Nunc Maxisorp™ flat bottomed 96 well plates. The wells were washed with PBS containing 0.05% Tween-20 and blocked with PBS containing 1% BSA for 30 min at room temperature. Supernatants of anti-WEE hybridoma cells or purified anti-WEE antibodies were then added to the wells and incubated for 1 hr at room temperature. The wells were subsequently washed and incubated with alkaline phosphatase-conjugated rat anti-mouse Ig antibody for 1 hr at room temperature. After washing, detection of the plates was performed by incubating the wells with phosphatase substrate solution for 30 min at 37° C. The isotypes of the antibodies were determined by identifying positive yellow reactions, corresponding to specific antibody isotypes. In addition, antibody isotypes were confirmed using alternate isotyping kits from Cedarlane Laboratories Ltd., Homby, Ontario and Gibco BRL.
Immunoprecipitation of WEE
WEE proteins were prepared in one-step in vitro transcription and translation reactions using the TNT® coupled system (Promega Corporation, Madison, Wis.). In the presence of rabbit reticulocyte lysate, transcription of the pCXH-3 plasmid resulted in WEE RNA. The pCXH-3 plasmid was constructed by cloning the entire WEE 26S region into pCI (Promega) at the XbaI and Sma I restriction sites. The WEE 26S region was placed under the control of the T7 RNA polymerase promoter and the cytomegalovirus enhancer/promoter (Netolitzky et al., 2000). The RNA was translated in the presence of [35S]-methionine to produce radiolabeled WEE proteins, which were further processed with canine pancreatic microsomal membranes. All components of the in vitro transcription and translation reactions were incubated together for 90 min at 30° C.
The TNT® reactions were diluted to a volume of 500 μl with RIP buffer consisting of 0.15 M sodium chloride, 0.1% SDS, 50 mM Tris-HCl pH 7.4, and 1% Triton X-100, and subjected to a preabsorption step by incubating with 75 μl of protein G-agarose (Gibco BRL) for 30 min at room temperature. The samples were centrifuged at 13,000×g for 1 min, and the supernatants were then incubated with either 100 μl of supernatants from anti-WEE hybridoma cells or 20 μg of purified anti-WEE antibodies. The reactions were incubated for 1.5 hr at room temperature, after which 75 μl of protein G-agarose was added. The reactions were incubated for an additional 30 min at room temperature. Immunoprecipitated proteins were collected by centrifuging at 13,000×g for 1 min. The pellets were washed with 500 μl of RIP buffer and centrifuged at 13,000×g for 1 min; this step was repeated three additional times. The pellets were resuspended in 2× Tricine sample buffer (Bio-Rad Laboratories) containing fresh 2% β-mercaptoethanol and heated at 100° C. for 10 min. The samples were centrifuged at 13,000×g for 1 min, and the supernatants were collected. The immunoprecipitated [35S]-labeled WEE proteins were further analyzed by SDS-PAGE and autoradiography. Radiolabeled [14C]-molecular weight markers from Amersham Pharmacia Biotech were also run on the polyacrylamide gels.
Preparation of Mouse Monoclonal Hybridoma Cell Lines and Purification of Antibodies
A total of 24 hybridoma cell lines which reacted with inactivated WEE antigen in ELISA assays were isolated. Of the 24 cell lines, 17 were chosen for further in-depth analysis (Table 1). A select number of anti-WEE antibodies (3F3E9G5, 3F6E3F8, 9B10D4D11G4, 10B5E7E2, 8F8D2F7E11, 11D2E11F2, and 11H9E2C12) were purified using protein G columns. In all purifications, concentrations of antibody were highest in fractions 3 and 4, ranging from 1 to 2 mg/ml.
Antigen Binding Activity of Antibodies
The present study first sought to determine the antigen binding activity of monoclonal antibodies using the indirect ELISA assay. WEE antigen (10 μg/ml) was immobilized onto 96 well plates and incubated with antibody. Absorbance values of controls, where no antigen was present, were subtracted from absorbance values for samples containing antigen. At various dilutions, each of the supernatants possessed antigen binding activity and displayed absorbance (405 nm) readings>0.154 (data not shown). The antigen binding activity of each of the antibodies is compared in Table 1, where the maximum dilutions of antibody supernatant used in ELISAs are listed. Certain antibodies (2B7C8G2, 3F3E9G5, 3F6E3F8, 5C5A5E5, and 10B5E7E2) showed strong reactivity at >1/320 dilutions, whereas other antibodies (1G6C1H5, 5C5B7H10, 5F11F2G11, and 10A7D10F5) showed weak reactivity to WEE at 1/20 dilutions. Absorbance readings were also taken with different antigen concentrations, at fixed concentrations of the 3F3E9G5 antibody (20 μg/ml) (
The study next sought to determine which WEE proteins were specifically recognized by each of the mouse monoclonal antibodies. Western blotting techniques found that a subset of antibodies were capable of detecting WEE proteins (
Immunoprecipitation experiments were performed to demonstrate that the antibodies were capable of binding to native WEE proteins. It was found that only a limited number of antibodies were capable of immunoprecipitating WEE proteins with protein G agarose (
Lastly, cross-reactivity experiments were performed in order to determine if the anti-WEE antibodies displayed binding activity to other alphaviruses. Antigen from SIN, VEE, or EEE was immobilized onto ELISA plates and incubated with antibody. Several antibodies, 3F3E9G5, 9B10D4D11G4, and 11D2E11F2, cross-reacted with SIN antigen, whereas other antibodies 2B7C8G2, 2D1E11F8, 5C5A1H11, and 11H9E2C12 reacted with EEE (Table 1). The 11H9E2C12 antibody bound not only to WEE and EEE, but also VEE.
Protection from WEE infection and disease are relevant and important issues affecting a large number of the population. It has been found that in mice, protection from alphavirus by activated T cells alone is not effective or sufficient. Instead, clearance and protection from infectious virus in the nervous system are accomplished by delivered antibodies (Griffin et al., 1997). Thus, an important method for protection against WEE may be facilitated by passive immunization, where viral-specific antibodies are administered to help prevent illness or mediate recovery of individuals exposed to virus. A limited number of monoclonal antibodies with both neutralization and passive immunization activity against alphaviruses have been previously found (Mathews and Roehrig, 1982; Boere et al., 1983; Yamamoto, 1986; Johnston and Peters, 1996; Griffin et al., 1997). The present study reports the construction and characterization of a collection of mouse monoclonal antibodies capable of recognizing WEE proteins for potential use in identification and therapy studies.
The 24 hybridomas expressing different anti-WEE antibodies are isolated, and the most reactive antibodies are evaluated further. The monoclonal antibodies all show varying reactivity to WEE in ELISAs, both with the supernatants and with the purified fractions. The antibodies 2B7C8G2, 3F3E9G5, 3F6E3F8, 5C5A5E5, and 10B5E7E2, display the highest binding activity to WEE, at dilutions >1/320. Others display binding activity at dilutions >1/100. Detection of WEE antigen by 3F3E9G5 is sensitive to less than 1 μg/ml when 20 μg/ml of antibody is used. These antibodies are strong candidates for use in ELISA based WEE detection assays. Of these antibodies listed, only 3F3E9G5 (E2), 3F6E3F8 (E1), and 10B5E7E2 (E2) are reactive against WEE protein in Western blotting. Furthermore, of these three antibodies, only 3F3E9G5 is reactive in immunoprecipitations. From these results, it appears that of the strongly binding WEE antibodies, 3F3E9G5 is the most versatile antibody, capable of recognizing WEE proteins in “native” and “denatured” forms and in a number of different assays.
Many of the antibodies, 1G6C1H5 (E1), 2D1E11F8 (NC), 3F3E9G5 (E2), 3F6E3F8 (E1), 5F11F2G11 (E1), 9B10D4D11G4 (E2), 10A7D10F5 (E1), 10B5E7E2 (E2), 11D2E11F2 (E1), and 11H9E2C12 (NC) display activity in Western blotting and recognize WEE proteins with clear resolution. The antibodies 3F3E9G5, 10A7D10F5, and 10H9E2C12 are not only capable of recognizing WEE proteins in Western blotting but also in immunoprecipitations, indicating that these three antibodies may be capable of recognizing E2, E1, and NC respectively in not only “denatured” but also “native” forms. 5C5A1H11 (E2) and 8F8D2F7E11 (E1) mayonlyrecognize WEE proteins in their “native” forms, as these antibodies are reactive only in ELISAs and immunoprecipitations but not in Western blotting.
The monoclonal antibody against western equine encephalitis virus, reference no. 8F8D2F7E11, has been deposited at the International Depositary Authority of Canada, National Microbiology Laboratory, Health Canada, at 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3R2 on Jan. 13, 2004 and was assigned Accession No. 120104-01.
A subgroup of the anti-WEE antibodies is also capable of binding to other alphavirus antigens. The antibodies 3F3E9G5, 9B10D4D11G4, and 11D2E11F2 bind to SIN, whereas 2B7C8G2, 2D1E11F8, and 5C5A1H11 bind to EEE. One antibody, 11H9E2C12, recognizes three different alphaviruses, WEE, VEE, and EEE. This is not entirely surprising as a large number of viruses in the alphavirus genus are closely related in terms of molecular characteristics and structure (Strauss and Strauss, 1994; Johnston and Peters, 1996). For instance, alphavirus nucleocapsids are antigenically similar. The nuclecapsid gene of WEE is closely related to the analogous regions of EEE (Hahn et al., 1988). Interestingly, 2D1E11F8 and 11H9E2C12, antibodies which recognize the nucleocapsid of WEE, bind to EEE. The E1 and E2 sequences of WEE are most closely aligned with comparable sequences of SIN (Hahn et al., 1988). This study also finds that 3F3E9G5, 9B10D4D11G4, and 11D2E11F2 bind to one of the WEE glycoproteins and is cross-reactive with SIN. Because the above antibodies recognize other alphaviruses in addition to WEE, the antibodies may potentially have functions in multiple systems, not only in WEE based assays but also in SIN, VEE, and EEE immunodetections and immunotherapies.
The information derived from the characterization of the mouse monoclonal antibodies may be used in further immunological studies. These antibodies can be used to detect WEE in a number of forms, as the antibodies have different specificities and reactivities in various assays. Recombinant antibodies such as scFv, Fab, and bispecific antibodies, can be constructed from each of the hybridoma clones expressing anti-WEE monoclonal antibodies. From the hybridoma expressing 10B5E7E2, a scFv retaining good recognition to the WEE antigen was constructed. This scFv was fused to the human IgG1 heavy chain to produce a chimeric antibody which may show potential for immunotherapy (Long et al., 2000). These recombinant antibodies along with the mouse monoclonal antibodies can serve in a wide range of applications, ranging from immunohistochemistry immunoassays, radioimmunodiagnosis, radioimmunotherapy, and immunotherapy (Hayden et al., 1997; Cao and Suresh, 1998).
It is to be understood that the embodiments and variations shown and described herein are merely illustrative of the principles of this invention and that various modifications may be implemented by those skilled in the art without departing from the scope and spirit of the invention.
The present application is a divisional of U.S. Ser. No. 09/793,606, filed Feb. 27, 2001, now U.S. Pat. No. 6,812,329.
| Number | Date | Country | |
|---|---|---|---|
| 20040229220 A1 | Nov 2004 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09793606 | Feb 2001 | US |
| Child | 10874370 | US |
