MX2007009512A - Neutralizing monoclonal antibodies against severe acute respiratory syndrome-associated coronavirus. - Google Patents

Abstract

The present invention provides an isolated antibody capable of binding to the receptor- binding domain of the spike protein of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) so as to competitively inhibit the binding of the SARS-CoV to host cells. These mAbs or substances can be used: 1) as passive-immunizing agents for prevention of SARS-CoV infection; 2) as biological reagents for diagnosis of SARS-CoV infection; 3) as immunotherapeutics for early treatment of SARS-CoV infection; and 4) as probes for studying the immunogenicity, antigenicity, structure, and function of the SARS- CoV S protein.

Description

NEUTRALIZATION OF MONOCLONAL ANTIBODIES AGAINST CORONAVIRUS ASSOCIATED WITH SEVERE ACUTE RESPIRATORY SYNDROME REFERENCE TO RELEVANT REQUESTS This application claims the benefit of the application of EUA Series No. 60 / 651,046, filed on February 8, 2005, and is a continuation in part of the US Application Serial No. 11 / 141,925, filed May 31, 2005, which claims the benefit of the US Application Serial No. 60 / 576,118, filed on June 2, 2004. References are made to various publications to through this application. Accordingly, these references and their corresponding disclosures are hereby incorporated by reference in their entireties in this application to provide a more complete description of the state of the branch to which this invention pertains. BACKGROUND OF THE INVENTION Severe acute respiratory syndrome (SARS) is a severe, febrile, recently recognized respiratory disease that is the result of an infection caused by a novel coronavirus (SARS-Co.V). (1-5). The global attack of SARS; It was contained, but there were still concerns about the possibility of future recurrences, especially with the recent reports of infections acquired in the laboratory (6). However, there is currently no effective treatment or prophylaxis available to combat this deadly virus (7.8). Like other antivirals, SARS-CoV is a enveloped virus that contains a large, positive-strand RNA genome that encodes viral replicase proteins and structural proteins that include pico (S), membrane (M), envelope (E), nucleocapsid (N), and several uncharacterized proteins (4, 5, 9). Filigenic analyzes indicate that SARS-CoV is distinct from three known antigen groups of coronaviruses. Therefore, the post-genomic characterization of SARS-CoV is important to develop therapeutic and anti-SARS vaccines (10, 11). The coronavirus infection is initiated by fixing the S protein to the specific host receptor, which triggers a conformational change in the S protein. The S protein of SARS-CoV is a type I transmembrane glycoprotein with a predichá length of 1,255 amino acids containing a leader (residues 1-14), an ectodomain (residues 15-1190), a transmembrane domain (residues 1191- 1227), and a short intracellular tail (residues 1227-1255) (5). Unlike many other counterviruses, such as mouse hepatitis virus (MHB) (12, 13), where protein S is post-translationally separated into SI and S2 subunits, no typical separation motif has been identified in the S-protein SAR-CoV (5). However, their SI and S2 domains were predicted by sequence alignment with other S proteins of coronaviruses (5, 14). The S2 domain (residues 681-1255) of SARS-CoV S protein containing a putative fusion peptide and two repeating heptad regions HR1 and HR2) is responsible for fusion between viral and target cell membranes. It has been found that the HR1 and HR2 regions can be associated to form a six-helix bundle structure (15-18), which resembles the active nucleus by fusion of HIV gp41 (19) and MHV S protein (20, 21). ). The SI domain of SARS-CoV S protein mediates the virus binding with angiotensin-converting enzyme 2 (ACE2), the functional receptor for SARS-CoV in susceptible cells (22-25). Recently, a small 193-amino acid fragment within the SI domain (residues 318-510) was identified as a receptor binding domain (RBD), which is sufficient to associate with ACE2 (26-28). The S proteins of coronavirus are major antigen determinants that induce the production of neutralization antibodies (29, 30). In this way, the use of S protein as an antigen for vaccine development follows logically (30). Recently, it has been shown that the SARS-CoV S protein is a major inducer of protective immunity between structural proteins (31). Yang et al. (32) reported that a candidate DNA vaccine encoding protein S induced neutralization of SARS-CoV (neutralization antibody titers ranged from 1:25 to 1: 150) and protective immunity in mice, and it was proved that the protection was mediated by neutralization antibodies but not by a T cell-dependent mechanism. Bisht, et al. (33) demonstrated that the SARS-CoV S protein expressed by 8MVA attenuated vaccine virus elucidated specific S antibodies with the titer of 1: 284 of SARS-CoV neutralizing antibody, and immunized mice in a protective manner against infection of SARS-CoV as shown by reduced titers of SARS-CoV in the respiratory tracts of mice after challenge infection. Bukreyev, et al. (34) reported that mucosal immunization of African green monkeys with an attenuated parainfluidin virus (BHPIV3) expressing the SARS-CoV S protein induced neutralization antibodies with neutralization titers ranging from 1: 8 to 1:16 and They protected animals against the challenge infection. These data indicate that the SARS-CoV S protein is a protective antigen capable of inducing neutralization antibodies, even though its antigenic determinants remain to be defined. We recently demonstrated that the SARS-CoV receptor S receptor binding (RBD) domain is a major target of neutralization antibodies induced in patients infected with SARS-CoV and in animals immunized with inactivated virus or S proteins (35, 36). Therefore, we use the recombinant RBD of the SARS-CoV S protein as an immunogen to induce monoclonal neutralizing antibodies (mAbs). BRIEF COMPENDIUM OF THE INVENTION The present invention provides an isolated monoclonal antibody capable of binding to the receptor binding domain (RBD) of the spike protein (S) of the coronavirus associated with severe acute respiratory syndrome (SARS-CoV) so as to competitively inhibit the binding of SARS-CoV to host cells. Additionally, the present invention provides a substance comprising the complementary determination regions of the monoclonal antibody described above, capable of binding to the same epitope as the monoclonal antibody described above. In one embodiment, the substance described above is an antibody. In a preferred embodiment, the antibody is neutralization. The present invention also provides a single chain antibody or antibody fusion construct; a humanized antibody; and a chimeric antibody as described above. It is the intention of the present application to cover different chimeric constructions created using the invented antibodies. The present invention also covers all humanized constructions of antibodies. In one embodiment, the isolated antibody described above is coupled directly or indirectly to cytotoxic agents. The present invention also provides cells comprising the antibody. The present invention additionally provides a nucleic acid molecule encoding the above antibody. The present invention further provides a nucleic acid molecule capable of specifically hybridizing the above-described molecule. The nucleic acid molecule includes, but is not limited to, synthetic DNA or RNA, genomic DNA, cDNA and RNA. The present invention also provides a vector comprising the above nucleic acid molecules or a portion thereof. In one embodiment, the vector is an expression vector, by which the protein encoded by the above nucleic acid molecules can be expressed. This invention further comprises a cell comprising the nucleic acid molecules described above. The cells can be used for expression. The present invention provides a method for producing the antibody capable of binding to the receptor binding domain (RBD) of the spine (S) protein of SARS-CoV in order to competitively inhibit the binding of SARS-CoV to host cells, which comprises operatively linking the nucleic acid molecule described above to the appropriate regulatory element so as to express the antibody; placing the bound nucleic molecule under appropriate conditions that allow the expression of the antibody; and recovering the expressed antibody, thereby producing the antibody. This invention also provides an antibody produced by the above method. The present invention provides a composition comprising an effective amount of the monoclonal antibody described above in an appropriate carrier. The present invention further provides a pharmaceutical composition comprising an effective amount of the above-described monoclonal antibody and a pharmaceutically acceptable carrier. The present invention also provides a method for treating SARS-CoV infection using the above pharmaceutical composition. The present invention further provides a method for preventing SARS-CoV infection using the above pharmaceutical composition. The present invention also provides a method for detecting SARS-CoV (or cells infected with SARS-CoV), which comprises contacting the antibody or its derivative capable of binding the receptor binding domain (RBD) of the spike protein ( S) of the virus under conditions that allow the formation of complexes between the antibody, or its derivative, and the RBD of SARS-CoV S protein; and detect the complexes formed. Finally, the present invention provides a method for screening compounds capable of inhibiting infection of coronaviruses associated with severe acute respiratory syndrome (SARS-CoV) by blocking the binding of the virus to receptors in host cells, comprising the steps of (a) establishing a system for the antibody to bind to the I receptor binding domain (RBD) of the spike protein (S) of the SARS-CoV; and (b) contacting the compounds with the system of (a), whereby a decrease in the binding of the above antibody to the RBD of SARS-CoV S protein indicates that the compounds are capable of interfering with the binding, inhibiting in this way the infection of the RBD of protein S of SARS-CoV. This invention also provides the resulting screened compounds. The compounds can then be used to treat or prevent severe acute respiratory syndrome (SARS). The present invention provides an equidype comprising a compartment that contains an antibody capable of recognizing the SARS virus. The present invention demonstrates that the receptor binding domain (RBD) contains multiple neutralization epitopes, conformation-dependent, that induce a panel of potent neutralizing monoclonal antibodies (mAbs), which can be used for the treatment, diagnosis and prevention of SARS. BRIEF DESCRIPTION OF THE DIVERSE VIEWS OF THE DRAWINGS Figure 1. Epitope mapping of mAbs 4D5 and 17H9 overlapping peptides covering the RBD of S protein. Each of the peptides was coated at 5 ug / ml and mAbs were tested at 10 ug / ml. ml. Figure 2. Inhibition of binding of RBD-Fc to ACE2 by mAbs. The upper panel shows the inhibition of RBD-Fc that binds to ACE2 associated with cell expressed in 293T / ACE2 cells measured by flow citodometry; the lower panel shows the inhibition of RBD-Fc which links to soluble ACE2 measured by ELIASA. RBD-Fc was used at 1 ug / ml and mAbs were used at 50 ug / ml. The percent inhibition was calculated for each mAb. Figure 3. Neutralization of SARS pseudovirus by mAbs. Inhibition of SARS pseudovirus infection in 293T / ACDE2 cells by mAbs representative of each group was shown. Each of the mAbs was tested in a series of 2-fold dilutions and the% neutralization was calculated. DETAILED DESCRIPTION OF THE INVENTION The present invention provides an isolated monoclonal antibody capable of binding to the receptor binding domain (RBD) of the spike protein (S) of the coronavirus associated with serevero acute respiratory syndrome (SARS-CoV) in order to competitively inhibit the binding of SARS-CoV to host cells. The present invention also provides a substance comprising the regions of complementary determination of the monoclonal antibody described above, capable of binding to the same epitope as the monoclonal antibody described above. This substance includes, but is not limited to, a polypeptide, small molecule, antibody, or a fragment of an antibody. In a preferred embodiment, the antibody is neutralization. In another embodiment, the antibody is a single chain antibody or antibody fusion construct; a humanized antibody; or a chimeric antibody as described above. It is the intention of this application to cover different chimeric constructions created using the invented antibodies. The present invention also covers all humanized constructions of antibodies. The branch of generating chimeric or humanized antibodies is well known, See, e.g., (37, 38) for chimeric antibodies and (39-41) for humanized antibodies. An ordinarily skilled artisan can modify the sequence of the substance described above in the light of the present disclosure. Such modification may include addition, omission, or mutation of certain amino acid sequences in the fragment. The general method for producing an antibody is within the knowledge of one of ordinary experience in the field. See, e.g., Using Antibodies: a Laboratory Manual: Portable Protocol No. 1 by Ed. Harlow (1998). In one embodiment, the isolated antibody described above is coupled directly or indirectly to one or more cytotoxic agents. Said cytotoxic agent includes, but is not limited to, radionucleotides or other toxins. The present invention also provides cells comprising the antibody. The present invention additionally provides a nucleic acid molecule encoding the above antibody. Once the antibodies are isolated, the gene encoding the antibody can be isolated and the nucleic acid sequence will be determined. Accordingly, the present invention further provides a nucleic acid molecule capable of specifically hydridizing the above-described molecule. The nucleic acid molecule includes, but is not limited to, synthetic DNA or RNA, genomic DNA, cDNA, and RNA. The present invention also provides a vector comprising the above nucleic acid molecules or a portion thereof. This portion may be a functional portion that performs a certain function. A fragment or partial sequence may be capable of encoding a functional domain of the protein that is functional. In one embodiment, this vector is an expression vector, by which the protein encoded by nucleic acid molecule can be expressed. The present invention further provides a cell comprising the nucleic acid molecule described above. Said cells can be used for expression. Vectors are well known in this field. See, eg, Graupner, U.S. Patent No. 6,337,208, "Cloning Vector", issued January 8, 2002. See also, Schumacher et al., U.S. Patent No. 6,190,906, "Vector Expression for the Regulable Expression of Foreign Genes in Prokaryotes ", issued February 20, 2001. In one embodiment, the vectors are plasmids. The present invention provides a method for producing the antibody capable of binding to the receptor binding domain (RBD) of the spike protein (S) of SARS-CoV in order to competitively inhibit the binding of SARS-CoV to host cells, which comprises, operatively linking the nucleic acid molecule described above to an appropriate regulatory element in order to express the antibody, placing the bound nucleic molecule under appropriate conditions that allow the expression of the antibody; and recovering the expressed antibody, thereby producing the antibody. The present invention also provides an antibody produced by the above method. The hybridoma cell lines 32H5 (Conf I), 31H12 (Conf II), 18D9 (Conf III), 30F9 (Conf IV), 33G4 (Conf V), and 19b2 (conf VI () were deposited on January 13, 2005 with the American Type Culture Collection 8ATCC9, 10801 University Blvd., Manassas, VA 20110, USA, under the provisions of the Budapest Treaty for the International Recognition of Microorganism Deposit for the purposes of Patent Procedure. (Conf I), 31H12 (Conf ii), 18D9 (Conf III), 30F9 (Conf IV), 33G4 (Conf V), and 19B2 (Conf VI) received the ATCC Accession Numbers PTA-6525, PTA-6524, PTA-6521, PTA-6523, PTA-6526, and PTA-6522, respectively The present invention also provides epitopes recognized by the monoclonal antibodies described above, said epitopes, sequence or conformation, are important for diagnostic and therapeutic uses. The present invention provides a composition comprising an effective amount of monoclonal antibody described above and an appropriate carrier. The effective amount can be determined by routine experimentation. The present invention further provides a pharmaceutical composition comprising an effective amount of the monoclonal antibody described above and a pharmaceutically acceptable carrier. As used herein, a pharmaceutically acceptable carrier means any of the conventional pharmaceutical carriers. Examples of suitable carriers are well known in the art and may include, but are not limited to, any of the conventional pharmaceutical carriers such as phosphate buffered saline solutions, Polysorb 80 containing phosphate buffered saline, water, emulsions such as emulsion. of oil / water, and various types of wetting agents. Other carriers may also include sterile solutions, tablets, coated tablets and capsules. Typically these carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, fats or vegetable oils, gums, glycols, or other known excipients. These carriers may also include flavor and color additives or other ingredients. The compositions comprising these carriers are formulated by conventional well-known methods. The present invention also provides a method for treating SARS-CoV infection using the above pharmaceutical composition. The present invention further provides a method for preventing infection of SARS-CoV using the above pharmaceutical composition. The present invention further provides a method for detecting SARS-CoV (or cells infected with SARS-CoV), comprising contacting the antibody, or its derivative, capable of binding to the receptor binding domain (RBD) of the protein of spike (S) of the virus under conditions that allow the formation of complexes between the antibody, or its derivative, and the RBD of the SARS-CoV S protein; and detect the complexes formed. Finally, the present invention provides a method for screening compounds capable of inhibiting SARS-CoV infection by blocking the binding of the virus to receptors in host cells, comprising the steps of (a) establishing a system for the antibody to bind to the domain receptor binding (RBD) of spike protein (S) from SARS-CoV; and (b) contacting the compounds with the system of (a), whereby a decrease in binding of the above antibody to the RBD of the SARS-CoV S protein indicates that the compounds are capable of interfering with the link, thus inhibiting the RBD infection of the SARS-CoV S protein. The present invention also provides the resulting sifted compounds, which can be used to treat or prevent severe acute respiratory syndrome (SARS). The present invention provides a kit comprising a compartment containing an antibody capable of recognizing the SARS virus and / or a substance that can competitively inhibit the binding! of the antibody. The present invention demonstrates that RBD contains multiple conformation-dependent neutralization epitopes that induce a panel of potent neutralizing monoclonal antibodies (mAbs), which can be used for the treatment, diagnosis and prevention of SARS. The invention will be better understood by reference to the following Experimental Details, but those skilled in the art will readily appreciate that the specific detailed experiments are illustrative only, and are not intended to limit the invention as described herein, which is defined by the claims that follow later. Experimental Details Twenty-seven hybridoma clones were generated by fusing SP2 / 0 myeloma cells with the splenocytes of Balb / c mice immunized with a fusion protein containing a binding domain, receptor (RBD) in spike protein (S) of SARS- CoV linked to fragment of human IgGl Fc (designated RBD-Fc). Among the 27 monoclonal antibodies (mAbs) prodecuous from these hydridome clones, except 2 mAbs linked to the adjacent linear epitopes, all the other mAbs recognized conformation-dependent epitopes. Based on the results obtained from the link competition experiments, these 25 conformation-specific mAbs could be divided into six groups, designated Conf I-VI. The Conf IV and Conf V mAbs blocked significantly in RBD-Fc binding to ACE2, the receptor for SARS-CoV, suggesting that their epitopes overlap with the receptor binding sites in the S protein. Most mAbs ( 23725) that recognized the formation epitopes possessed potent neutralizing activities against SARS pseudovirus with 50% neutralization dose (ND50) ranging from 0.005 to 6,569 ug / ml. These SARS-CoV neutralization mAbs can be used: 1) as immunotherapeutics for early treatment of SARS-CoV infection; 2) as biological reagents for diagnosis of SARS-CoV infection; and 3) as probes to study the immunogenicity, antigenicity, structure and function of the SAR-CoV S protein. In addition, these murine mAbs can be humanized for therapy and prevention of SARS-CoV infection. Materials and methods Immunization of mice and generation of mAbs. Five Balb / c mice (4 weeks old) were immunized subcutaneously with 20 ug of purified RBD-Fc with Protein A Sepharose prepared as described above (35) in the presence of MLP + TDM Adjuvant System (Sigma, Saint Louis, MI ) and was increased with 10 ug of the same antigen plus the MLP + TDM adjuvant at 3-week intervals. Mouse antisera were collected to detect anti-RBD antibodies and neutralization antibodies of SARS-CoV. Hydrobromomes to produce anti-RBD mAbs were generated using conventional protocol. Briefly, splenocytes from idmmunized mice were harvested and fused with SP2 / 0 myeloma cells. Cell culture supernatants from wells containing hybridoma colonies were screened by enzyme-linked immunosorbent assay (ELISA) using S1-C9 prepared as described above 835). as a coating antigen. Positive well cells expanded and retested. Cultures that remained positive were subcloned to generate stable hybridoma cell lines. All mAbs were purified from the culture supernatants by Protein A Sepharose 4 Fast Flow (Amersham Biosciences). ELISA and liaison competition. The reactivity of sera from mice or mAbs with various antigens was determined by ELISA. Briefly, 1 ug / ml of recombinant proteins (RBD-Fc or S1-C9) or purified human IgG (Zymed, South San Francisco, CA) were used, respectively, to coat 96-well microtiter plates (Corning Costar, Acton, MA) in 0.1 M carbonate buffer (pH 9.6) at 4 ° C overnight. After blocking with 2% nonfat milk, serially diluted mouse sera or mAbs were added and incubated at 37 ° C for 1 h, followed by four washes with PBS containing 0.1% Tween 20. The bound antibodies were detected with goat anti-mouse IgG conjugated goat (Zymed) at 37 ° C for 1 hour, followed by washings. The reaction was visualized by addition of the substrate 3, 3 ', 5, 5' -tetramethylbenzidine (TMB) and absorption at 450 nm was measured by an ELISA plate reader (Tecan US, Research Triangle Park, NC). To determine the effect of disulfide bond reduction on the binding of RBD-specific mAbs, the ELISA plate was coated with recombinant RBD-Fc or S1-C9 at a concentration of 3 1 ug / ml and then treated for 1 hour at 37 ° C with dithiothreitol (DTT) at a concentration of 10 M, followed by washes. Then the wells were treated with 50 mM of iodoacetamide for 1 h at 37 ° C. After washing, a conventional ELISA was performed as described above. A competitive ELISA was performed to determine the inhibitory activity of the RBD-specific mAbs of the biotinylated mAbs to RBD-Fc. Briefly, the wells of ELISA plates were coated with RBD-Fc at 1 ug / ml as described above. A mixture containing 50ug / ml of an unlabelled mAb and 1ug / ml of a biotinylated mAb was added, followed by incubation at 37 ° C for 1 hour. The linkage of the thiobinylated mAbs was detected after addition of streptavidin conjugated with HRP (Zymed) and TMB in sequence. Biotinylation of mAbs was performed using the EZ-lin, NHS-PEO Solid PCER Biotinylation Kit (Pierce, Rockford, IL) in accordance with the manufacturer's protocol. Neutralization of SARS pseudovirus infection.

The conventional neutralization test using SARS-CoV vido is annoying and has to be performed in BSL-2 facilities. The SARS-CoV S protein containing SARS pseudovirus and a defective HIV-1 genome expressing luciferase as a reporter was prepared as described previously (27, 42, 43). Briefly, 293T cells were transfected with a plasmid coding for codon-optimized SARS-CoV S protein and a plasmid encoding Env-defective, HIV-1 I genome expressing luciferase (pNL4-3, luc.RE) using 6 Fugene reagents (Boehringer Mannheim). Supernatants containing SARS pseudovirus were harvested 48 hours after transfection and used for single-cycle infection of 293T cells transfected with ACE2 (2935 / ACE2). Briefly, 293% / ACE2 cells were plated at 104 cells / well in 96-well tissue culture plates and developed overnight. Supernatants containing pseudoviruses were preincubated with mouse sera serially diluted 2-fold or mAbs at 37 ° C for 1 hour before addition to cells. The culture was fed back with fresh medium 24 and incubated for an additional 48 hours. The cells were washed with PBS and used using a lysis reagent included in a luciferace kit (Promega, Madison, Wl). Aliquots of cell lysates were transferred to 96-well Costar flat bottom Luminometer plates (Corning Costar, Corning, NY), followed by the addition of luciferaza substrate (Promega). Relative light units (RLU) were determined immediately in the Ultra 384 luminometer (Tecan US). Inhibition of RBD-FC binding with receptor by mAbs. Inhibition of mAbs in RBD-Fc that bind to cells expressing ACE2 was measured by flow cytometry. Briefly, 106 293T / ACE2 cells were separated, harvested and washed with Hank's balanced saline solution (HBSS) (Sigma, St. Lou7is, MO). The RBD-Fc was added to the cells at a final concentration of 1 ug / ml, in the presence or absence of 50 ug / ml of mAbs, followed by incubation at room temperature for 30 min. The cells were washed with HBSS and incubated with anti-human IgG-FITC conjugate (Zymed) at 1:50 dilution at room temperature for an additional 30 min. After washing, cells were fixed with 1% formaldehyde in PBS and analyzed on a Becton FACSCalubir flow cytometer (Mountain View, CA) using CellQuest software. The inhibition of RBD-Fc that binds soluble ACE2 by mAbs was measured by ELISA. Briefly, the recombinant soluble ACE2 (R & amp; amp;; D systems, Inc., Minneapolis, MN) at 7 ug / ml was coated in 96-well ELISA plates (Corning Costar) in 0.1 M carbonate buffer (pH 9.6) at 4 ° C overnight. After blocking with 2% non-fat milk, 1 μg / ml of BRD-Fc was added to the wells in the presence or absence of 50 μg / ml of mouse mAbs and incubated at 37 ° C for 1 hour. After washing, goat anti-human IgG conjugated with HR4P (Zymed) was added and incubated for an additional hour. After washing, the TMB substrate was used for detection. Results Isolation and initial characterization of specific mAbs for RBD. The RBD-Fc fusion protein was expressed transiently in 293T cells and purified to homogenicity by Protein A. Five mice (A to E) were immunized four times with RBD-Fc in the presence of Ribi adjuvant. All animals developed notorious antibody responses against FBD-Fc after the first increase, and their antibody titers increased with subsequent immunizations. The antisera collected 4 days after the third increase showed highly potent neutralizing activity against SARS-CoV and SARS pseudovirus containing SARS-CoV S protein. A panel of 27 mAbs specific for RBDE were generated by fusing splenocytes from mice immunized with RBD-Fc with Sp2 / 0 myeloma cells and then screening hybridomas with S1-C9 as an antigen. The epitope specificities of these mAbs were initially determined by ELISAs using RBD-Fc, reduced RBD-Fc with DTT, S1-CÍ9, YES-C9 reduced with DTT, a human IgG purified as coating antigens / Table I). The majority of the mAbs (25/27) were reactive with native RBD-Fc and S1-C9, but not RBD-Fc reduced with DTT and S1-C9. This' indicated that they were directed against disulfide-binding-dependent conformation epitopes expressed in the S-protein BRD. Two other mAbs (4D5 and 17H9) recognized both native RBD-Fc and Si-C9 as reduced, indicating that they were directed against epitopes linear presented in the RBD. None of the mAbs screened by S1-C9 reacted with human IgG, while the control antiserum of a mouse immunized with RBD-Fc was reactive with human IgG (Table I). Since mAbs 4D5 and 17H9 were able to react with reduced RBD-Fc and S1-CÍ9, their epitopes could be mapped with synthetic peptides. A set of 27 overlapping peptides covering the RBD of protein S was used to locate 4D5 and 17H9 epitopes by ELISA. As shown in Figure 1, 4D5 reaction with peptide 435-451 (NY6NYKYRYLRHGKLRPF), and 17H9 reacted with two overlapping peptides 442-458 (YLRHGKLRPFERDISNV) and 449-465 (RPFERDISVNVPFSPDGK). While the epitope of 17H9 was clearly mapped to the overlapping sequence (RPFERDIS V) of peptides 442-458 and 449-465, the epitope for 4D5 requires the majority of peptide sequence 435-451 that overlaps partial sequences of peptides 442 -458 and 449-465. Therefore, these two mAbs recognize neighboring linear epitopes that reside within the RBDE. None of the conformation-dependent mAbs reacted with any of the treated peptides (data not shown). Epitope specificity of the RBD-speci fi c mAbs determined by binding competition assays.

In order to characterize the conformation-dependent epitopes, the RBD-specific mAbs were pooled by binding competition assays (Table II). One of the mAbs (10E7) was first biotinylated and the inhibitory activity of the 27 mAbs in 10E7 that binds to RBD-Fc was measured. Mabs 4D5 and 17H9 recognizing linear epitopes mapped by the above peptides were included in the competition assays as a control. About half of the conformation-dependent mAbs (13/25) competed with biotinylated 10E7, while other mAbs did not block 10E7 that binds to RBD-Fc. Four other of the non-competent mAbs (111 E12, 33G4, 45B5, and 17H9) were subsequently biotinylated and tested in a similar manner with the binding competition assay. Five of the 13 mAbs that competed with the biotinylated 10E7 also blocked the biotinylated 45B5 that binds to RBD-Fc and were designated as a separate group. In this way, the 25 conformation-specific mAbs were divided into six distinct competence groups (designated Conf I-VI). Two linear epitope-specific mAbs (4D5 and 17H9) did not compete with any of the specific confounding mAbs. These results suggest that the RBD of protein S contains multiple antigenic structures that induce specific antibody responses in the mice. However, the immunodominant epitopes in the RBD are conformation dependent. Characterization of mAbs blocking receptor link. RBD-Fc could be efficiently ligated to ACE2 expressed in 293T / ACE2 cells and soluble ACE2 as measured by flow cytometry and ELISA, respectively (data not shown). We test whether the specific RBD mAbs inhibit the binding of RBD-Fc to associated cell or soluble ACE2. As shown in Figure 2, all Conf IV mAbs (28D6, 39F9, and 35B5) and Conf V (24F4, 33G4 and 38D4) completely blocked RBD-Fc which binds to both cell-associated ACE2 and soluble in a highly consistent way. All of the two Conf III mAbs (11E112 and 18D9) and two of the four Conf V mAbs (19B2 and 45F6) partially inhibited BRD-Fc which binds to ACE2 expressed in 293T / AEC2 and soluble ACE2 cells. All other mAbs, including two mAbs against linear sequences, had no significant inhibitory effects on receptor binding. These results indicate that the Conf IV and Conf V mAbs recognize epitopes that can overlap with the conformation receptor binding sites on the S protein, even though these mAbs did not compete against each other in the binding competition assays. Conf III mAbs and two Conf VI mAb 819B2 and 45F6) are also linked to the conformational epitopes that are involved in the receptor binding. All Conf I and Conf II mAbs did not block receptor binding, suggesting that they recognize conformation epitopes that do not overlap the receptor binding sites in RBD. These results highlight the epitope heterogeneity of RBD-specific mAbs and further indicate that the RBD of S protein contains multiple antigenic conformations. Specific mAbs of RBD have potent neutralization activity. Each of the specific RBD mAbs was tested for neutralization activity against SARS pseudovirus. Surprisingly, the majority of the conformation dependent mAbs (23/25) had potent neutralization activity with 50% neutralization dose (ND50) ranging from 0.005 to 6,569 ug / ml (Table III), while two mAbs that were direct linear epitoids (4D5 and 17H9) and a Mab of Conf VI (44B5) at a concentration as high as 100 ug / ml did not neutralize the infection of SARS pseudovirus. The Conf V and 30F9 mAbs 33G4 of Conf IV that blocked the receptor linkage had the highest neutralization activities against the pseudovirus. Interestingly, still 45F6 of Conf VI, with its relatively lower pseudovirus neutralization activity, partially blocked the link of RBD-Fc with ACE2. The dose-dependent neutralization activity of several representative mAbs from each of the groups was presented in Figure 3. These results suggest that the RBD of S protein predominantly induces neutralizing antibodies that are directed against conformational epitopes. Experimental Discussion Recent studies have shown that the SARS-CoV S protein is one of the major antigens that produce immune responses during infection (44-46). These suggest that S protein can serve as an immunogen to induce neutralization of mAbs. In the present study, we used a recombinant fusion protein RBD-Fc as an immunogen to immunize mice and the hybridoma clones generated to produce 27 mAbs. A majority of these mAbs (25/27) recognized conformational epitopes and among them, 23 mAbs had potent neutralizing activity. Only two mAbs were mapped to adjacent linear epitopes by overlapping peptides and failed to neutralize infection by SARS pseudovirus. Interestingly, the conformation-dependent mAbs could be divided into six different groups (ie, Conf I-VI) based on the binding competition experiment, suggesting that there are several distinct conformational epitopes in the RBD that can produce antibodies of neutralization. It is expected that all neutralization mAbs directed against RBD can block the interaction between RBD and ACE2, the functional receptor for SARS-CoV. However, we found that only mAbs that recognize Conf IV and V could effectively block RBD to ACE2. Some mAbs that react with Conf III and VI partially inhibited the interaction between RBD and ACE2. This suggests that their epitopes may overlap the receptor binding sites in the RBD or the binding of these RBD mAbs may cause conformational change of the receptor binding sites, resulting in inhibition of RBD that binds to ACDE2. Mabs recognizing Conf I and 1 (1 did not significantly affect RBD binding with ACE2, but also possessed potent neutralizing activities, suggesting that these mAbs inhibit SARS-Cov infection without interfering with the interaction of RBD-ACE2. The mechanism of action of these mAbs needs further investigation.These data indicate that RBD reduces the neutralization antibodies specific not only to the receptor binding sites, but also to other unique structural conformations, accentuating their antigenic heterogeneity, and suggest that the RBD of SARS-CoV S protein contains multiple conformation epitopes responsible for the induction of potent neutralizing antibody responses.The conformational sensitivity of the mAbs that neutralize SARS-CoV described herein is consistent with properties of neutralizing mAbs against other enveloped viruses , which generally require more native conformation for linkage (47, 48) Even though the RBD of the SARS-CoV S protein is a small fragment of 193 amino acids, it contains seven cysteines and five of which are essential for association of ACE2. The disulfide bonds between these cysteines can form complex tertiary structures to form the multiple antigenic conformations. However, a human neutralization mAb selected from a non-immune human antibody library was able to react with the reduced S protein with DTT and block the receptor association (49). Therefore, further characterization is needed to define the neutralizing determinants in the RBD of SARS-CoV S protein, and this may provide critical information for developing SARS therapeutics and vaccines. It was reported that passive transfer of mouse immune sera reduced viral viral replication in mice challenged with SARS-CoV (33, 50), and prophylactic administration of neutralization mAbs conferred protection in vivo in mice or in ferrets ( 51, 52), suggesting passive immunization with antibodies against SARS is a viable strategy for controlling SARS. In this way, mAbs with high levels of SARS-CoV neutralization activity can be used for the early treatment of SARS-CoV infection. However, the application of murine MAbs in human will be limited due to human-anti-mouse antibody (HAMA) responses (53-55). If only a few doses of murine mAbs are used in a short period of time (one or two weeks) in the early stage of SARS-CoV infection, it may not cause serious HAMA, but this urgent treatment can save lives of SARS patients. We have used similar strategies for the early treatment of Hantaan virus (HTNV) infection using murine anti-HTNV mAbs 856). In addition, murine neutralization mAbs can be humanized as therapeutic or immunoprophylaxis to provide immediate protection against SARS-CoV infection to those populations at risk. The importance of the present study is threefold. First, the number of highly potent specific RBD neutralization mAbs has been generated, which can develop immunotherapeutics for the urgent treatment of SARS. Second, these mAbs can be developed as diagnostic agents to detect SARS-CoV infection. Third, these mAbs can be used as probes to study the immunogenicity, antigenicity, structure and function of the SARS-CoV S protein. These mAbs can be further humanized for treatment and prevention of SARS. Table I. Reactivities of specific RBD mAbs against various antigens3 mAbs Isotype Antigen RBD-Fc FBD-Fc S1-C9 YES-C9 Reduced Human IgG Reduced 4D5 IgGl / k 0.88 1.36 0.65 094 0.02 9F7 IgGl / k 1.60 0.00 1.45 0.08 0.04 10E7 IgGl / k 1.77 0.02 1.72 0.16 0.05 11E12 IgG2a / k 1.50 0.01 0.72 0.09 0.02 12B11 IgGl / k 1.37 0.05 0.78 0.00 -0.01 13B6 IgGl / k 1.58 -0.01 0.93 0.00 0.02 17H9 IgGl / k 1.72 1.71 1.21 1.15 0.07 18C2 IgGl / k 1.28 -0.01 0.80 0.01 -0.20 18D9 IgGl / k 1.47 -0.01 0.90 0.01 0.03 19B2 IgGl / k 1.63 0.00 1.55 0.12 0.01 20E7 IgG2a / k 1.50 0.00 0.98 0.01 0.02 24F4 IgGl / k 1.69 -0.01 1.08 0.08 0.04 24H8 IgGl / k 1.54 -0.01 0.94 0.12 0.01 26A4 IgGl / k 1.60 0.00 0.89 0.09 0.01 26E1 IgGl / k 1.91 0.07 1.85 0.06 0.01 27CF1 IgGl / k 1.46 0.00 1.57 0.07 0.01 28D6 IgGl / k 2.08 0.01 1.60 0.16 0.00 29G2 IgG2a / k 1.69 0.00 0.96 0.17 0.01 30F9 IgGl / k 1.66 0.04 1.21 0.12 0.03 31H12 IgGl / k 1.72 0.08 1.91 0.22 0.03 32H5 IgGl / k 1.54 0.06 1.55 0.51 0.00 33G4 IgG2a / k 1.79 0.02 1.76 0.20 -0.01 34E10 IgGl / k 1.62 0.10 1.82 0.18 0.04 35B5 IgGl / k 1.74 0.06 1.72 0.25 0.02 38D4 IgGl / k 1.63 -0.01: 1.20 0.07 0.00 44B5 IgGl / k 1.57 0.09 1.64 0.16 0.00 45F6 IgGl / k 1.61 0.11 1.43 0.15 -0.01 Antiserum 2.22 1.78 2.32 1.68 2.07 Naive Serum 0.01 0.02 0.02 0.01 0.04 a Antigens were used at 7 ug / ml; mAbs were tested at 10 ug / ml and the sera were tested at a dilution of 3: 1: 100. Positive reactivities are marked in dark Table II. % inhibition of RBD-specific mAbs when ligating thiotinylated mAbs to RBD-Fc. Biotinylated mAb mAb group Competition 10 E7 11 E12 33G4 45B5 17H9 Conf I 9F7 84.5 11.7 -13.3 22.3 16.4 10E7 91.0 5.6 -12.9 21.0 9.9 12B11 85.8 19.3 -0.2 19.8 21.0 18C2 84.9 19.3 4.9 18.1 19.4 24H8 93.7 24.0 7.0 25.6 22.1 26E1 95.1 10.5 37.4 30.4 25.0 29G2 96.6 20., 4 1.6 11.4 23.5 32H5 98.9 18.5 4.4 9.1 20.3 Conf II 20E7 97.2 38.5 5.9 73.0 24.6 26A4 96.3 33.1 -0.5 60.0 19.0 27C1 97.2 36.7 14.6 73.7 20.9 31H12 97.6 18.7 7.1 58.4 19.7 30E10 98.3 19.3 12.9 68.9 24.6 Conf III 11E12 12.6 92.0 0.3 -3.7 20.2 18D9 -16.2 98.3 8.3 23.6 17.1 Conf IV 28D6 39.7 99.6 13.8 67.4 26.6 30F9 28.7 100.0 8.7 64.0 32.4 35B5 34.9 99.9 10.0 64.7 33.6 Conf V 24F4 11.5 -1.0 95.5 2.5 24.9 33G4 9.5 -3.7 99.5 26.4 29.1 38D4 8.1 -14.4 82.0 -5.1 15.8 Conf VI 13B6 23.3 10.7 -4.0 72.5 12.6 19B2 2.9 -26.4 18.0 50.0 16.1 44B5 25.3 -20.6 10.0 95.6 19.4 45F6 25.7 -10.4 10.8 94.8 23.5 Linear 4D5 13.9 10.6 -11.1 1.0 -10.5 17H9 17.8 33.3 -5.8 25.0 97.8 a Competing mAbs were tested at 100 ug / ml for the ability to block the binding of biotinylated mAbs to RBD-Fc in ELI8SA. More than 40% inhibition is considered positive competence (dark values). Negative numbers indicate increased binding of the biotinylated reagent. Table III. Neutralization activity of RBD specific mAbs against pseudovirus SARS Mab group Inhibition of ACE2a ND50 binding (ug / ml) Conf I 9F7 - 6,569 10E7 - 1,673 12B11 - 4,918 18C2 - 5,031 24H8 - 3,955 26E1 - 0.354 29G2 - 3.02 32H5 - 0.275 Conf II 26A4 - 2.815 27C1 - 1.607 31H12 - 0.139 30E10 - 0.399 Conf III 11E12 + 1.39 18D9 + 0.02 Conf IV 28D6 ++ 0.298 30F9 ++ '0.009 35B5 ++ 0.131 Conf V 24F4 ++ 0.052 33G4 ++ 0.005 38D4 ++ 0.332 Conf VI 13B6 - 1.436 19B2 + 0.936 44B5 - > 100 45F6 + 43,894 Linear 4D5 - > 100 17H9 - > 10Q to "_v ^" + "and" ++ "indicate no inhibition, partial and complete, respectively. References 1. Drosten, C, S. Gunther, W. Preiser, W.S. Go Der, H.R. Brodt, S. Becker, H. Rabenau, M. Panning, L.

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Claims (19)

1. An isolated antibody with binding capacity to the spike protein receptor domain of the coronavirus associated with severe acute respiratory syndrome (SARS-CoV) or antibody with the ability to competitively inhibit the binding of the coronavirus associated with severe acute respiratory syndrome ( SARS-CoV) to a receptor on the host cells or to a cell-free receptor.
2. A substance or antibody comprising the complementarity determining regions of the antibody of claim 1, capable of binding to the same epitope or competitively inhibiting the binding of the epitope as the antibody of claim 1.
3. The antibody of claim 1, characterized in that the antibody is produced by hybridoma 19D9 with accession number ATCC PTA-6521, hybridoma 19B2 with accession number ATCC PTA-6522, hybridoma 30F9 with accession number ATCC PA-6523, hybridoma 31H12 with accession number ATCC PTA-6524, hybridoma 32H5 with accession number ATCC PTA-6525 or hybridoma 33G4 with accession number ATCC PTA-, 6526.
4. . The epitope recognizing the antibody of claim 3.
5. 5. The antibody of claim 1, characterized in that the antibody is a single-chain antibody, a humanized antibody, a chimeric antibody, a neutralizing antibody or a fusion construction of the antibody.
6. 6. The isolated antibody of claim 1, characterized in that the antibody is coupled directly or indirectly to a cytotoxic agent.
7. 7. A cell containing the antibody of claim 1.
8. 8. A nucleic acid molecule encoding the antibody of claim 1, characterized in that the nucleic acid molecule is a synthetic DNA, a genomic DNA, cDNA or RNA.
9. A nucleic acid molecule: capable of specifically hybridizing the molecule of claim 8.
10. 10. A vector or cell comprising the nucleic acid molecule of claim 8 or a part thereof.
11. 1. A method for producing an antibody capable of binding to the receptor domain of the spike protein of the coronavirus associated with severe acute respiratory syndrome (SARS-CoV) or the antibody capable of competitively inhibiting the binding of the coronavirus associated with acute respiratory syndrome Severe (SARS-CoV) to the host cells, which consists in operatively ligating the nucleic acid molecule of claim 8 to the appropriate regulatory element to express the antibody; placing the bound nucleic molecule under appropriate conditions that allow the expression of the antibody; and recovering the expressed antibody, to thereby produce the antibody.
12. 12. The antibody produced by the method of claim 11.
13. 13. A composition containing an effective amount of the antibody of claim 1 and an appropriate carrier.
14. 14. A pharmaceutical composition containing an effective amount of the antibody of claim 1 and a carrier accepted for pharmaceutical use. I
15. 15. The use of the antibody of claim 1, in the treatment or prevention of infection of the coronavirus associated with severe acute respiratory syndrome (SARS-CoV), or in the manufacture of treatment to treat or prevent infection of the coronavirus associated with acute respiratory syndrome serious (SARS-CoV).
16. 16. A method for detecting the coronavirus associated with severe acute respiratory syndrome (SARS-CoV) or cells infected with SARS-CoV, which consists of contacting an antibody or its derivative capable of binding to the receptor binding domain of the spike protein from the virus under conditions that allow the formation of complexes between the antibody, or its derivative, and the receptor-binding domain of the spike protein of the coronavirus associated with severe acute respiratory syndrome (SARS-CoV); and detect the complexes formed.
17. 17. A method for screening compounds capable of inhibiting infection of the coronavirus associated with severe respiratory syndrome by blocking the binding of the virus to the receptors on the host cells, consists of the steps of: '(a) establishing a system for the antibody of the claim 1 for binding to the spike protein receptor binding domain of the coronavirus associated with severe acute respiratory syndrome (SARS-CoV); and (b) contacting the compounds with the system of (a), a decrease in binding of the antibody of claim 1 to the spike protein receptor domain of the coronavirus associated with severe acute respiratory syndrome (SARS-CoV) indicating that the compounds are capable of interfering with the binding and inhibiting infection of the spike protein receptor domain of the coronavirus associated with severe acute respiratory syndrome (SARS-CoV).
18. 18. The compound resulting from the method of claim 17.
19. 19. A kit containing a compartment containing the antibody of claim 1.