MONOCLONAL ANTIBODY PRODUCTION BY EBV TRANSFORMATION OF B CELLS

Abstract

A method for producing a clone of an immortalised human B memory lymphocyte, comprising the step of transforming human B memory lymphocytes using Epstein Barr virus (EBV) in the presence of a polyclonal B cell activator. The method is particularly useful in a method for producing a clone of an immortalised human B memory lymphocyte capable of producing a human monoclonal antibody with a desired antigen specificity, comprising the steps of: (i) selecting and isolating a human memory B lymphocyte subpopulation; (ii) transforming the subpopulation with Epstein Barr virus (EBV) in the presence of a polyclonal B cell activator; (iii) screening the culture supernatant for antigen specificity; and (iv) isolating an immortalised human B memory lymphocyte clone capable of producing a human monoclonal antibody having the desired antigen specificity.

Claims

1. A method for preparing a recombinant cell, the method comprising: (i) preparing an immortalized B cell clone, comprising transforming a human memory B lymphocyte using Epstein Barr virus (EBV) in the presence of a polyclonal B cell activator, wherein the polyclonal B cell activator is an agonist of a Pattern Recognition Receptor that is expressed on human memory B lymphocytes; (ii) obtaining, from the immortalized B cell clone, a nucleic acid molecule that encodes an antibody of interest; and (iii) inserting the nucleic acid molecule encoding the antibody of interest into an expression host cell, thereby preparing a recombinant cell.

2. The method of claim 1, wherein the expression host cell is a yeast cell, a plant cell, or an animal cell.

3. The method of claim 1, wherein the nucleic acid molecule encoding the antibody of interest is modified to: (a) introduce one or more restriction site into the nucleic acid molecule; (b) change usage of one or more codon in the nucleic acid molecule; (c) introduce one or more transcription regulatory sequence into the nucleic acid molecule; (d) introduce one or more translation regulatory sequence into the nucleic acid molecule; or (e) any combination of (a)-(d).

4. The method of claim 1, wherein the Pattern Recognition Receptor comprises a Toll Like Receptor (TLR) that is expressed on memory B cells.

5. The method of claim 4, wherein the TLR is Toll Like Receptor 7 (TLR7), Toll Like Receptor 9 (TLR9) or Toll Like Receptor 10 (TLR10).

6. The method of claim 5, wherein the agonist of the Pattern Recognition Receptor is CpG 2006 (SEQ ID NO:1).

7. A method for preparing a nucleic acid molecule, the method comprising: (i) preparing an immortalized B cell clone, comprising transforming a human memory B lymphocyte using Epstein Barr virus (EBV) in the presence of a polyclonal B cell activator, wherein the polyclonal B cell activator is an agonist of a Pattern Recognition Receptor that is expressed on human memory B lymphocytes; (ii) sequencing, from the immortalized B cell clone, a nucleic acid molecule that encodes an antibody of interest; and (iii) using the sequence information from (ii) to prepare a nucleic acid molecule for insertion into an expression host cell in order to permit expression of the antibody of interest by the expression host cell, thereby preparing the nucleic acid molecule.

8. The method of claim 7, wherein the nucleic acid molecule encoding the antibody of interest is modified to: (a) introduce one or more restriction site into the nucleic acid molecule; (b) change usage of one or more codon in the nucleic acid molecule; (c) introduce one or more transcription regulatory sequence into the nucleic acid molecule; (d) introduce one or more translation regulatory sequence into the nucleic acid molecule; or (e) any combination of (a)-(d).

9. A method for preparing a nucleic acid molecule that encodes an antibody of interest, the method comprising: (i) immortalizing human memory B lymphocytes, wherein the immortalizing comprises transforming human memory B lymphocytes using Epstein Barr virus (EBV) in the presence of a polyclonal B cell activator, wherein the polyclonal B cell activator is an agonist of a Pattern Recognition Receptor that is expressed on human memory B lymphocytes; (ii) isolating an immortalized B cell clone capable of producing an antibody of interest; and (iii) obtaining a nucleic acid molecule, from the immortalized B cell clone, that encodes the antibody of interest.

10. The method of claim 9, further comprising: (iv) using the nucleic acid molecule to prepare an expression host cell capable of expressing the antibody of interest; and (v) culturing or sub-culturing the expression host cell under conditions where the antibody of interest is expressed.

11. The method of claim 10, further comprising: (vi) purifying the antibody of interest.

12. The method of claim 10, wherein using the nucleic acid molecule to prepare the expression host cell in (iv) comprises inserting the nucleic acid molecule into the expression host cell.

13. The method of claim 10, wherein using the nucleic acid molecule to prepare the expression host cell in (iv) comprises: (a) obtaining sequence information of the nucleic acid molecule encoding the antibody of interest; and (b) using the sequence information to prepare a nucleic acid molecule encoding the antibody of interest for inserting into the expression host cell.

14. The method of claim 10, wherein the nucleic acid molecule encoding the antibody of interest is modified to: (a) introduce one or more restriction site into the nucleic acid molecule; (b) change usage of one or more codon in the nucleic acid molecule; (c) introduce one or more transcription regulatory sequence into the nucleic acid molecule; (d) introduce one or more translation regulatory sequence into the nucleic acid molecule; or (e) any combination of (a)-(d).

15. A method for obtaining a nucleic acid sequence that encodes an antibody of interest, the method comprising: (i) immortalizing human memory B lymphocytes, wherein the immortalizing comprises transforming human memory B lymphocytes using Epstein Barr virus (EBV) in the presence of a polyclonal B cell activator, wherein the polyclonal B cell activator is an agonist of a Pattern Recognition Receptor that is expressed on human memory B lymphocytes; (ii) isolating an immortalized B cell clone capable of producing an antibody of interest; and (ii) obtaining sequence information, of a nucleic acid molecule encoding the antibody of interest, from the immortalized B cell clone.

16. The method of claim 10, wherein the antibody of interest is directed against respiratory syncytial virus (RSV), human immunodeficiency virus; hepatitis A virus; hepatitis B virus; hepatitis C virus; herpes simplex virus type I or type 2; SARS coronavirus; measles virus; mumps virus; rubella virus; rabies virus; ebola virus; influenza virus; papillomavirus; vaccinia virus; varicella-zoster virus; variola virus; polio virus; rhino virus; a human endogenous retrovirus; P. falciparum; P. vivax; P. malariae; P. ovale; Corynebacterium diphtherias; Clostridium tetani; Clostridium botulinum; Bordetella pertussis; Haemophilus influenzae; Neisseria meningitidis; serogroup A, B, C, W135 and/or Y; Streptococcus pneumoniae; Streptococcus agalactiae; Streptococcus pyogenes; Staphylococcus aureus; Bacillus anthracis; Moraxella catarrhalis; Chlamydia trachomatis; Chlamydia pneumoniae; Yersinia pestis; Francisella tularensis; Salmonella species; Vibrio cholerae; toxic E. coli; TNF-?, ?-amyloid protein, SARS coronavirus spike protein, prion protein PrP, complement C5, CBL, CD147, IL-8, HIV glycoprotein gp120, VLA-4, CDI Ia, CD18, VEGF, CD40L, an ICAM, a VCAM, CD80, TPL2, Her2 or an integrin.

17. A method of treating or preventing, in a subject, a condition characterized by expression of: serogroup A, B, C, W135 and/or Y; TNF-?; ?-amyloid protein; SARS coronavirus spike protein; prion protein PrP; complement C5; CBL; CD147; IL-8; HIV glycoprotein gp120; VLA-4; CDI Ia; CD18; VEGF; CD40L; an ICAM; a VCAM; CD80; TPL2; Her2; or an integrin, the method comprising administering to the subject an antibody prepared according to the method of claim 16.

18. A method of treating or preventing, in a subject, an infection by: respiratory syncytial virus (RSV); human immunodeficiency virus; hepatitis A virus; hepatitis B virus; hepatitis C virus; herpes simplex virus type I or type 2; SARS coronavirus; measles virus; mumps virus; rubella virus; rabies virus; ebola virus; influenza virus; papillomavirus; vaccinia virus; varicella-zoster virus; variola virus; polio virus; rhino virus; a human endogenous retrovirus; P. falciparum; P. vivax; P. malariae; P. ovale; Corynebacterium diphtherias; Clostridium tetani; Clostridium botulinum; Bordetella pertussis; Haemophilus influenzae; Neisseria meningitidis; Streptococcus pneumoniae; Streptococcus agalactiae; Streptococcus pyogenes; Staphylococcus aureus; Bacillus anthracis; Moraxella catarrhalis; Chlamydia trachomatis; Chlamydia pneumoniae; Yersinia pestis; Francisella tularensis; Salmonella species; Vibrio cholerae; or toxic E. coli, the method comprising administering to the subject an antibody prepared according to the method of claim 16.

19. A method of detecting RSV in a subject, the method comprising contacting a biological sample from the subject with an anti-RSV antibody prepared according to the method of claim 16, wherein the anti-RSV antibody is detectably labeled.

20. A method of activating an isolated human B memory lymphocyte, the method comprising contacting an isolated human B memory lymphocyte with an agonist of a Pattern Recognition Receptor (PRR) expressed on memory B cells.

21. The method of claim 20, wherein the PRR comprises a Toll-Like Receptor (TLR) which is expressed on memory B cells.

22. The method of claim 21, wherein the agonist of the PRR comprises one or more of: (i) CpG (SEQ ID NO:1); (ii) R-848 (iii) imidazoquinoline compounds that stimulate TLRs; (iv) CD40L; (v) BAFF; (vi) a cell that expresses CD40L or BAFF; (vi) a monoclonal antibody that mimics the effect of a polyclonal B cell activator; or (vii) any combination of (i)-(vi).

Description

BRIEF DESCRIPTION OF DRAWINGS

[0105] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0106] FIG. 1 shows results of the ELISA using SARS virus-infected Vero cells lysed in 3% SDS as antigen. Shown are the OD values of serum (1/5000 dilution), supernatants of polyclonal cultures and of independent B cell clones (1/2 dilution).

[0107] FIG. 2 shows the neutralizing titer of antibodies specific for the SARS virus. From left to right: neutralization titer (Vero cell assay) of convalescent serum; supernatants of polyclonal cultures; positive clones isolated from the culture with highest neutralizing titer.

[0108] FIGS. 3A and 3B show clonal analysis of the human antibody response to the SARS virus spike protein. Culture supernatants were tested for their capacity to stain BHK cells transfected with SARS virus spike protein mRNA (Frankfurt isolate) and for their capacity to neutralize the same strain of SARS virus. FIG. 3A shows the correlation between neutralizing titer and staining of spike protein by undiluted culture supernatant. FIG. 3B shows staining of spike-transfected BHK cells by serial dilutions of supernatants from eleven neutralizing cultures, with filled symbols showing the maximum dilution where complete neutralization was observed.

[0109] FIG. 4 shows the characterization of the SARS neutralizing antibody S3.1. Staining of spike-transfected BHK cells by purified S3.1 (circles) and 6 months convalescent serum (squares). The filled symbols indicate the maximum dilution where complete neutralization was observed.

[0110] FIGS. 5A-5H shows immunoelectron microscopy of SARS coronavirus in the presence of S3.1 antibody.

[0111] FIG. 6 is an overview of a method of the invention, from human to monoclonal antibody.

MODES FOR CARRYING OUT THE INVENTION

[0112] The present invention permits the cloning of human memory B lymphocytes with very high efficiency and achieves this by the combination of two stimuli, namely: EBV, that immortalizes human B cells with low efficiency and a polyclonal B cell activator that enhances the efficiency of EBV-immortalization.

Example 1: Cloning of B Cells

[0113] Human memory B cells (CD19.sup.+ CD27.sup.+ IgM.sup.? IgD.sup.?) were isolated from healthy donors by cell sorting using methods well known in the art. Different numbers of cells were seeded in replicate cultures in 96 well microplates in the presence of irradiated mononuclear cells (5?10.sup.5/ml) and either EBV (supernatant of B95-8 cells) alone or EBV in combination with CpG 2006 (2.5 ?g/ml) and recombinant IL-2 (1000 U/ml). After 15 days the percentage of cultures containing growing cells was scored. Frequencies were determined by limiting dilution assays. Cultures were scored for growing cells. Cloning efficiency using four different sources of B cells were as follows:

TABLE-US-00001 Cloning efficiency B cell source EBV EBV + CpG + IL-2 Exp 1 (CD19.sup.+ CD27.sup.+ IgM.sup.? IgD.sup.?) 1 in 200 1 in 1 Exp 2 (CD19.sup.+ CD27.sup.+ IgM.sup.? IgD.sup.?) 1 in 120 1 in 1.5 Exp 3 (CD19.sup.+ CD27.sup.+ IgM.sup.? IgD.sup.?) 1 in 60 1 in 1 Exp 4 (CD19.sup.+ CD27.sup.+) 1 in 90 1 in 1.6

[0114] No growth was observed in the absence of EBV.

[0115] Thus the methods of the invention allow virtually every human memory B cell to be cloned (efficiency close to 100%). The method is also suitable for subcloning.

Example 2: Production of Antibodies with a Desired Specificity

[0116] In a further experiment it was demonstrated that the immortalisation can be used to exploit immunological memory to produce human monoclonal antibodies of the desired specificity.

[0117] Mononuclear cells were isolated from 20 ml peripheral blood obtained from a healthy blood donor. CD19.sup.+CD27.sup.+IgG.sup.+ human memory B cells were isolated by cell sorting and seeded at 10 cells/well in 96 well microplates in the presence of EBV, CpG 2006 (2.5 ?g/ml), recombinant IL-2 (1000 U/ml) and irradiated mononuclear cells (5?105/ml). Seeding only 10 cells per well helps to increase cloning efficiency. After 15 days all cultures contained growing cells. A sample of supernatant was collected and tested in ELISA for total IgG antibodies and for IgG specific for Toxoplasma gondii, tetanus toxoid and measles virus. The supernatants were also tested in a measles virus neutralization assay using Vero cells as targets.

[0118] Some of the positive cultures identified were subcloned by limiting dilution to isolate specific clones producing the desired monoclonal antibody. Cultures were subcloned at 0.5 cells/well in the presence of CpG 2006 (2.5 ?g/ml), IL-2 (1000 U/ml) and irradiated PBMC (5?10.sup.5/ml).

TABLE-US-00002 Antibody Positive Specific clones isolated/ (detection method) cultures .sup.# attempts made * IgG (ELISA) 180/180 Anti-Toxoplasma gondii (ELISA) 23/180 5/6 Anti-tetanus toxoid (ELISA) 19/180 5/6 Anti-Measles virus (ELISA) 36/180 7/9 Anti-Measles virus (neutralization) 7/180 4/4 .sup.# For ELISA, number of cultures with OD > 0.8 in an assay with background <0.2; for neutralisation assay, number with complete protection from cytolitic effect of measles virus * Number of cases where at least one antigen-specific clone could be isolated, relative to the number of original cultures that were cloned. Cloning efficiency varied from 50 to 100%.

[0119] EBV-transformed B cell clones that produce IgG antibodies to measles virus, tetanus toxoid and Toxoplasma gondii could thus be isolated, showing that human monoclonal antibodies with multiple memory specificities can be prepared from a small sample of human peripheral blood.

Example 3: Immortalised Memory B Cells that Express Antibodies Specific for SARS Coronavirus

[0120] Blood samples were obtained from two patients with a clinical record of SARS. Both patients had serum anti-SARS antibodies as detected by two assays: (i) a neutralization assay which detects neutralizing antibodies directed against surface proteins of the SARS virus, likely the spike protein and (ii) an ELISA assay, that detects antibodies binding to any denatured protein of the SARS virus.

[0121] For the neutralization assay, serial dilutions of serum obtained from the blood were added to microplate wells containing Vero cells, followed by titrated amounts of SARS virus. After 2 days, the cytopathic effect was recorded by visual inspection. A conventional ELISA was also developed using SARS virus infected Vero cells lysed in 3% SDS as the antigen.

[0122] For the production of the clone of B cells producing monoclonal antibodies specific for the SARS virus, blood from the patient showing the higher titer of binding and neutralizing antibodies was selected.

[0123] Memory B cells carrying surface IgG were isolated using anti-human IgG microbeads, incubated with EBV (50% supernatant of B95-8 cells) for 6 hours and were then plated at 10 cells/well in 96 well microplates in the presence of 2.5 ?g/ml CpG 2006 and allogeneic irradiated PBMC (in these experiments IL-2 was omitted). After two weeks the culture supernatants were screened for the presence of specific antibodies.

[0124] Out of 1042 culture supernatants tested, 165 scored positive in the ELISA assay (FIG. 1). 23 of these cultures were cloned as above and specific clones were isolated from 16 of them. Some of the human monoclonal antibodies isolated recognise the nucleoprotein of the SARS virus in western blots, while some do not, suggesting that they may recognise different viral proteins (data not shown). None of these antibodies showed neutralizing activity.

[0125] Out of 1042 culture supernatants tested in the neutralization assay, seven showed low level neutralizing activity while two (A11 and D8) showed high neutralizing titer (1/512 and 1/256 respectively). The A11 culture was cloned by limiting dilution in the presence of CpG and irradiated PBMC and several B cell clones with comparably high neutralizing activity were isolated (FIG. 2). The A11 antibodies did not bind in the ELISA assay, but stained surface spikes of the SARS virus as detected by electron microscopy (data not shown).

[0126] Therefore, using this method it is possible to produce neutralizing antibodies specific for an antigen using only a small blood sample (around 10 ml) within a short timespan (30-40 days). The method also allows selection of the best antibody from a large pool, and is therefore a high throughput method.

[0127] The anti-SARS antibodies neutralise the SARS virus at concentrations of ?5 ng/ml. Neutralization of respiratory syncytial virus (RSV, a common cause of respiratory tract infections, especially in children) by commercially available humanized antibodies produced by conventional techniques requires around a 1000-fold higher antibody concentration (Johanson et al. 1997). Therefore the fully human antibodies produced by the method of the invention appear to be around 1000-fold more effective. This suggests that very small amounts of the antibody described here should be sufficient to prevent or cure SARS infection.

Example 4: Screening SARS Virus Convalescent Patients for Antibodies

[0128] Peripheral blood was obtained from a patient at different times after acute infection with SARS virus (2, 4 and 6 months after infection). PBMC were isolated by gradient centrifugation. IgG.sup.+ memory B cells were isolated by an improved method that avoids triggering of the B cell receptor. Total B cells were isolated from PBMC using CD22 microbeads (Miltenyi), which were found to be even better than using CD19 microbeads. The cells were stained with antibodies to human IgM, IgD and IgA and negative cells carrying surface IgG were isolated by cell sorting. B cells were pulsed with EBV (50% supernatant of B-95-8 cells) for 8 hours and then plated at 10 cells/well in 96 well U-bottom microplates in complete RPMI medium supplemented with 10% FCS, 2.5 ?g/ml CpG 2006 and irradiated PBMC (2?10.sup.5/ml). In this experiment IL-2 was not used. After 2 weeks the culture supernatants were screened for the presence of specific antibodies using the three assays described below. Positive cultures were cloned by limiting dilution in the presence of CpG 2006 and irradiated PBMC as above. Positive clones were expanded and the antibody produced was purified from culture supernatants by affinity chromatography on protein A columns (Amersham).

[0129] The Frankfurt isolate of the SARS virus (Genbank accession number AY310120) was used for three in vitro assays:

[0130] ELISA

[0131] Vero cells were infected at a multiplicity of infection of 0.01 plaque forming units per cell. Cell culture supernatant collected after 2 days was cleared by centrifugation at 3000 rpm, 5 min. The supernatant was subjected to centrifugation at 20,000 rpm for 2 hours in a Beckman SW28 rotor through a 20% sucrose cushion. The pellet was purified using a potassium tartrate/glycerol gradient and resuspended in 500 ?l THE buffer (10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 2 mM EDTA) to a protein concentration of approx 0.5 mg/ml. The antigen suspension used for the ELISA assay was prepared by adding 1% SDS to the viral pellet followed by boiling for 10 min. ELISA plates were coated with a 1:1000 dilution of SARS virus antigen in 0.1 M phosphate buffer. Dilutions of sera or culture supernatant were added and specific IgG1 antibodies were detected using alkaline phosphatase goat anti-human IgG. Results were expressed in arbitrary units (AU) relative to the 2 month sample (=1000AU). Sera from 20 normal donors tested were negative (<1 AU).

[0132] Staining

[0133] Antibodies specific for native spike protein of SARS virus were detected by flow cytometry. Briefly, the SARS virus spike gene was cloned in an appropriate vector and mRNA was transcribed in vitro and used to transfect BHK cells by electroporation. Transfectants were incubated with culture supernatants or serum, washed and stained with APC-labelled goat anti-human IgG antibody. This assay detects IgG1 antibodies directed against native spike antigen, most of which have neutralizing activity. Results were expressed in arbitrary units (AU) relative to the 2 month sample (=1000AU). Sera from 20 normal donors tested were negative (<1 AU).

[0134] In Vitro Neutralization

[0135] Sera or culture supernatants were diluted in log 2 steps and mixed with 75 TCID.sub.50 SARS virus in 25 ?l (virus titer was determined according to the method of Karber). The mixture was incubated for 45 min at room temperature. Subsequently, 50 ?l trypsinized Vero cells (1.5?10.sup.5 per ml) were added and incubated for 3 days at 37? C. and finally, the neutralization titer was determined by visual inspection to give the serum dilution that neutralizes 75 TCID.sub.50 SARS virus. The assays were performed in a biosafety level 4 laboratory.

[0136] Results were as follows:

TABLE-US-00003 Anti-SARS antibody detected by Months after infection ELISA Spike staining Neutralization 2 1000 1000 1/128 4 650 700 1/128 6 300 400 1/128

[0137] While normal sera were negative, the patient's serum collected at different time points after the onset of acute disease scored positive in the three assays. Antibodies detected by ELISA and those staining spike-transfected cells were highest 2 months after infection and decreased to about one third by six months. In contrast neutralizing antibodies remained constant with a titer of 1/128. The isotype of the antibodies detected in the Spike-binding and ELISA assays was exclusively IgG1, no IgA or IgM antibodies being detected (data not shown). Thus the post-infection serum of this person had moderate titers of neutralizing antibodies to the SARS virus and IgG antibodies that bound spike proteins and detected denatured antigens in ELISA.

Example 5: Kinetics and Frequencies of Specific Memory B Cells

[0138] IgG.sup.+ memory B lymphocytes from the 2-month, 4-month and 6-month post-SARS sera were immortalized with EBV under conditions where the number of B cells per culture was limiting, as described above (10 B cells per well). This strategy allows analysis of the product of only a few memory B cells per culture, thus ensuring that the specific antibody detected in positive cultures is monoclonal and, at the same time, increasing the probability of isolating a clone producing the desired antibody by limiting dilution. After two weeks of culture in the presence of EBV, CpG 2006 and irradiated feeder cells the culture supernatants were screened for the presence of specific IgG antibodies using ELISA or staining of spike transfectants. The frequency of cultures screening positive in the SARS virus ELISA assay or staining SARS virus spike transfectants were as follows:

TABLE-US-00004 Positive cultures/total cultures screened (%) Months after infection ELISA Spike staining 2 275/480 (57.3%) Not determined 4 123/480 (25.6%) 12/576 (2.1%) 6 44/480 (9/2%) 21/768 (2.7%)

[0139] The frequency of cultures producing antibodies detected by the ELISA assay was very high 2 months after infection and decreased by 4 and 6 months. The frequency of cultures producing antibodies against native spike protein measured at 4 and 6 months was lower. Importantly, the culture that scored positive for ELISA antibodies were distinct from those containing antibodies to the spike indicating that the two assays detect distinct non-overlapping antibody specificities. Furthermore, a sizeable proportion of IgG.sup.+ memory B cells are specific for the spike protein.

[0140] Tests were then carried out to see whether there is a correlation between spike binding and neutralizing activity. 56 culture supernatants which stained spike-transfected cells were tested for their capacity to neutralize the same SARS virus isolate from which the spike protein was cloned (FIG. 3A). Although the antibodies with the highest staining showed high neutralizing titers, there were some antibodies that neutralized efficiently in spite of poor staining while others stained spike transfectants, but failed to neutralize. Furthermore, when 11 supernatants with high neutralizing titer were analyzed, no clear correlation between staining and neutralization was evident (FIG. 3B). Taken together these results indicate that at the clonal level the response to spike is heterogeneous and that not all the anti-spike antibodies produced in the course of the natural infection are capable of neutralizing the virus.

Example 6: Isolation of Monoclonal Antibodies to SARS Virus

[0141] The results shown above prove that it is possible to interrogate the human B cell memory repertoire with a variety of assays to identify cultures producing an antibody of the desired specificity. In these experiments, 29 of 38 attempts (76%) at cloning positive cultures led to the isolation of one or more clones producing antibodies of the selected specificity. The EBV clones were stable and monoclonal antibodies were recovered in the culture supernatant at concentrations of 10-20 ?g/ml. Of these 29, 21 were positive in the ELISA assay and 8 were both positive in the spike staining assay and were able to neutralize SARS virus.

[0142] Out of the 21 independent clones that scored positive in the ELISA assay, 13 (62%) produced antibodies specific for the SARS virus nucleoprotein (NP) as detected by Western blot, while 5 did not recognize NP, but stained SARS virus infected cells, and the remaining 3 reacted only in the ELISA assay. As expected, none of these antibodies showed neutralizing activity.

[0143] Out of the 8 independent clones staining spike transfectants and neutralizing SARS virus, one (S3.1, IgG1?) was selected for in vivo neutralization assays. The monoclonal antibody from this clone was purified from the culture supernatant and tested for its capacity to stain spike-transfected cells and to neutralize SARS virus (FIG. 4). S3.1 neutralized 75 TCID.sub.50 SARS virus at concentrations of ?300 ng/ml, and was up to 300 fold more potent than convalescent serum. Furthermore S3.1 neutralized the Frankfurt and Urbani isolates with the same efficiency (data not shown), and decorated the spikes of SARS-CoV as detected by immunoelectron microscopy (FIG. 5).

Example 7: S3.1 Neutralizes SARS Infection in an Animal Model

[0144] The in vivo neutralizing activity of the S3.1 monoclonal antibody was tested in a mouse model of acute SARS infection. Purified antibody was transferred to na?ve mice by intraperitoneal injection to determine whether antibody alone could prevent replication of SARS virus in the respiratory tract.

[0145] Drs. L. J. Anderson and T. G. Ksiazek from the Centers for Disease Control and Prevention (CDC), Atlanta, Ga., provided SARS virus (Urbani strain) for use in an in vivo neutralization assay. The virus was isolated and passaged twice in Vero E6 cells at the CDC and was passaged in Vero cells for two additional passages in our laboratory to generate a virus stock with a titer of 10.sup.6.5 TCID.sub.50/ml. The Vero cells were maintained in OptiPro SFM (Invitrogen). A11 work with infectious virus was performed inside a biosafety cabinet, in a biosafety containment level 3 facility and personnel wore powered air purifying respirators (3M HEPA AirMate, Saint Paul, Minn.). The mouse studies were approved by the NTH Animal Care and Use Committee and were carried out in an approved animal biosafety level 3 facility. A11 personnel entering the facility wore powered air purifying respirators.

[0146] Four-to-six week-old female BALB/c mice purchased from Taconic (Germantown, N.Y.) were housed, 4 mice per cage. On day 0 mice, lightly anesthetized with isoflurane, were injected intraperitoneally with 3 different doses (800, 200, 50 ?g) of S3.1 antibody in 500 ?l or with the same volume of a polyclonal human Ig that lacks neutralizing activity. 24 hours later mice were intranasally challenged with 10.sup.4 TCID.sub.50 of SARS coronavirus. After two additional days mice were sacrificed and their lungs and nasal turbinates were removed and homogenized in a 5% w/v suspension in Leibovitz 15 medium (Invitrogen). Tissue samples were assessed for infection, and virus titers were determined as described above. Virus titers were expressed as log.sub.10 TCID.sub.50 per gram of tissue:

TABLE-US-00005 Virus replication in challenged mice Lungs Nasal turbinates Number Mean (?SE) Number Mean (?SE) Antibody infected virus titer infected virus titer S3.1 800 ?g 0/4 ?1.5 ? 0 * 2/4 2.5 ? 0.47 S3.1 200 ?g 0/4 ?1.5 ? 0 * 4/4 3.4 ? 0.41 S3.1 50 ?g 2/4 .sup.3.2 ? 1.36 4/4 4.8 ? 0.75 Control 800 ?g 4/4 .sup.7.5 ? 0.1 4/4 6.4 ? 0.41 * The lower limit of detection of infectious virus in a 10% w/v suspension of lung homogenate was 1.5 log.sub.10TCID.sub.50/gm and in 5% w/v suspension of nasal turbinates was 1.8 log.sub.10TCID.sub.50/gm. These values thus indicate no detectable virus.

[0147] Mice that received S3.1 mAb were thus protected from replication of challenge virus, particularly in the lower respiratory tract. The differences in viral titers when compared to the control were statistically significantly (p<0.05) in a Student's t-test. Significant restriction of virus replication in the upper respiratory tract was noted in the mice which received the highest dose of S3.1 mAb.

Example 8: R-848

[0148] R-848 is an agonist of TLR7 and TLR8. This compound was compared with CpG 2006 in terms of efficiency of EBV-induced immortalization of human B cells. Memory B cells were isolated from healthy donors using anti-CD19 or anti-CD22 magnetic microbeads followed by negative depletion of cells carrying IgM, IgD and IgA (or IgG). 48 replicate cultures were set up in 96 well U-bottomed microplates by limiting dilution at 30, 10 and 3 B cells per well in complete medium in the presence of irradiated mononuclear cells, EBV (20% supernatant from B95-8 cells) and in the presence or absence of 2.5 ?g/ml CpG 2006 or 2.5 ?g/ml R-848. The frequency of cultures positive for cell growth and Ig production was measured after 14 days and the efficiency of transformation was calculated. Results were as follows:

TABLE-US-00006 B cell source EBV +CpG 2006 +R848 Donor AOS CD19.sup.+ IgM.sup.? IgD.sup.? IgA.sup.? 1 in 150 1 in 3.5 1 in 2.5 Donor AOS CD22.sup.+ IgM.sup.? IgD.sup.? IgA.sup.? 1 in 300 1 in 2 1 in 1.5 Donor ASC CD19.sup.+ IgM.sup.? IgD.sup.? IgA.sup.? 1 in 320 1 in 2 1 in 2.4 Donor ASC CD19.sup.+ IgM.sup.? IgD.sup.? IgG.sup.? 1 in 280 1 in 4 1 in 2.2 Donor ETR CD22.sup.+ IgM.sup.? IgD.sup.? 1 in 230 1 in 1.9 1 in 2

[0149] R-848 and CpG 2006 are thus comparable in their capacity to increase the efficiency of EBV-induced immortalization. In addition, R-848 was comparable to CpG 2006 in the capacity to increase the cloning efficiency of polyclonal EBV-immortalized B cell lines. In the presence of R-848 the cloning efficiency of EBV cell lines ranged from 25 to 100% in 10 independent experiments.

Example 9: Isolation of High Affinity Antibodies Neutralizing SARS-CoV

[0150] A new series of monoclonal antibodies with SARS-CoV neutralizing capacity was produced as described above from immortalized memory B cells isolated from a convalescent patient six months after infection. Serial dilutions of supernatants from the B cell clones were tested for their antigen specificity (NP, matrix (M) or spike proteins), and their capacity to neutralize the cytopathic effect of SARS CoV (Frankfurt isolate) on Vero cells. The concentration of monoclonal IgG was measured by ELISA in the same culture supernatants. Neutralizing titers were expressed as the final concentration of IgG (ng IgG per ml) in tissue culture capable of completely neutralizing the virus (mean values of at least three tests). Results were as follows:

TABLE-US-00007 B cell clone Isotype Specificity Neutralizing titer S18.1 IgG, ? NP S20.1 IgG, ? NP S21.1 IgG, ? NP S23.4 IgG, ? NP S24.1 IgG, ? NP S13.1 IgG, ? Not determined S5.1 IgG, ? M S3.1 IgG, ? Spike 300 S101.1 IgG, ? Spike 40 S102.1 IgG, ? Spike 850 S103.3 IgG, ? Spike 350 S104.1 IgG, ? Spike 150 S105.2 IgG, ? Spike 150 S106.1 IgG, ? Spike 45 S107.4 IgG, ? Spike 75 S108.1 IgG, ? Spike 40 S109.2 IgG, ? Spike 80 S132.9 IgG, ? Spike 200 S128.5 IgG, ? Spike 25 S127.6 IgG, ? Spike 40 S124.4 IgG, ? Spike 40 S159.1 IgG, ? Spike 25 S160.1 IgG, ? Spike 15

[0151] Thus the invention is routinely able to provide antibodies that can neutralize the virus at concentrations lower than 10.sup.?9 M and even down to 10.sup.?10 M (MW of human IgG is ?150 kDa, and so 150 ng/ml is ?10.sup.?9 M).

[0152] It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention. In particular, minor modifications that do not affect the immunogenicity of the modified capsular saccharide of the present invention are also encompassed.

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