# Patent

## Title
Monoclonal antibodies specific for anthrax spores and peptides derived from the antibodies thereof

## Abstract

The present invention provides monoclonal antibodies which are highly specific for Bacillus spores. Also provided are peptides derived from those monoclonal antibodies. Both the antibodies and peptides are highly specific and can discriminate between spores of potentially lethal organisms such as Bacillus anthracis and other harmless but closely related bacilli and provide a very powerful tool in the construction of detection instruments as counter measures.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS 
The present application is a continuation of, and claims priority to, U.S. application Ser. No. 11/171,518, filed Jul. 1, 2005 now abandoned, which is a continuation of and claims priority to U.S. application Ser. No. 09/069,628, now U.S. Pat. No. 6,913,756, filed Apr. 29, 1998, which applications are incorporated herein by reference in their entirety.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with government support under GM 054068 awarded by the National Institutes of Health. The government has certain rights in the invention.


BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of immunology and microbiology. More specifically, the present invention relates to monoclonal antibodies specific for anthrax spores and peptides derived from the antibodies.
2. Description of the Related Art
During the evolution of the immune system there is evidence that the repertoire of germline V genes that has been retained in the genome has been subject to selective processes by environmental influences which may include self as well as commensal and non-commensal microorganisms. Structural and functional analysis of immunoglobulin and T cell receptors have delineated regions of these molecules which are germline encoded and have the ability to bind to certain bacterial components through exposed parts of the molecules which do not need somatic diversification for expression of the ability to bind to these structures. Some of these included protein A binding to framework three (FR3) region of VH genes, staphylococcal enterotoxin binding to T cell receptor, etc.
The prior art is deficient in the lack of monoclonal antibodies which are highly specific and can discriminate between spores of the Bacillus family including the strategically important B. anthracis. Further, the prior art is deficient in the lack of peptides derived from the monoclonal antibodies highly specific for Bacillus spores. The present invention fulfills this longstanding need and desire in the art.
SUMMARY OF THE INVENTION
The present invention is directed to monoclonal antibodies which are highly specific for anthrax spores and peptides derived from the antibodies. The present invention demonstrated that the humoral immune response to spores of Bacillus show a remarkable conservation of VH gene usage which is distinct for each spore analyzed. The results imply evolutionary conservation of VH genes due to their ability to bind spores. Furthermore, of highly practical importance, these antibodies can discriminate between spores of potentially lethal organisms such as B. anthracis and other harmless but closely related bacilli and provide a very powerful tool in the construction of detection instruments as counter measures in biological warfare.
In one embodiment of the present invention, there is provided a monoclonal antibody specific for Bacillus spores. Preferably, Bacillus is selected from the group consisting of Bacillus anthracis, Bacillus thuringiensis, Bacillus subtilis and other bacilli related to Bacillus anthracis. Preferably, the antibody is IgG.
In another embodiment of the present invention, there is provided a peptide derived from the monoclonal antibody highly specific for Bacillus spores.
In yet another embodiment of the present invention, there is provided a method of preparing the monoclonal antibody highly specific for Bacillus spores by immunizing and fusing local lymph nodes of an animal.
In still yet another embodiment of the present invention, there is provided a method of detecting Bacillus spores in a field sample using a monoclonal antibody highly specific for the Bacillus spores by contacting the sample with a monoclonal antibody disclosed herein.
In still yet another embodiment of the present invention, there is provided a method of detecting Bacillus spores in a field sample using a peptide derived from a monoclonal antibody highly specific for the Bacillus spores by contacting the sample with a peptide derived from a monoclonal antibody highly specific for Bacillus spores.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.



BRIEF DESCRIPTION OF THE DRAWINGS
So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions of the invention briefly summarized above may be had by reference to certain embodiments thereof which are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope.
FIG. 1 shows antibodies with different reactivities for germinated and ungerminated spores among different clones.
FIG. 2 shows that anti-spore monoclonal antibodies do not react with vegetative bacteria.
FIG. 3 shows that most antibodies react specifically with spores of Bacillus subtilis. 
FIG. 4 shows that a commonly used member VH7183.6 heavy chain gene of the VH7183 (MOPC21) family in all hybridomas reactive with Bacillus subtilis spores appears to be the most unique member of this family in the framework three (FR3) region. (Hybridomas g07 to f10 are labeled SEQ ID NO: 7 to SEQ ID NO: 17, respectively)
FIG. 5 shows that antibody-derived peptides specifically bind Bacillus subtilis spores.
FIG. 6 shows that anti-Bacillus anthracis antibody specifically bind Bacillus anthracis spores.
FIG. 7 shows that the VH gene sequences among monoclonal antibodies to Bacillus anthracis. (SEQ ID NO: 18 to SEQ ID NO: 31)
FIG. 8 shows the discriminatory ability of the antibodies in vitro. Bacillus anthracis, Bacillus subtilis and Bacillus thuringiensis spores were mixed and stained on a slide with fluorescent antibodies labeled with blue, green and red antibodies, respectively.
FIG. 9 shows the discriminatory ability of the antibodies in vivo. A section of mouse spleen was injected 30 minutes previously with Bacillus anthracis, Bacillus thuringiensis, Bacillus subtilis spores and labeled with blue, green and red antibodies, respectively.



DETAILED DESCRIPTION OF THE INVENTION
In the present invention, panels of monoclonal antibodies which are highly specific and can discriminate between spores of the Bacillus family including the strategically important Bacillus anthracis (anthrax) were isolated and characterized. The amino acid sequences of these anti-spore antibodies were determined from the nucleotide sequences of the coding genes and smaller peptide molecules were derived from these antibodies which can also bind Bacillus spores.
The present invention is directed to a monoclonal antibody highly specific for Bacillus spores. Preferably, Bacillus is selected from the group consisting of Bacillus anthracis, Bacillus thuringiensis, Bacillus subtilis and other bacilli closely related to Bacillus anthracis. Preferably, the antibody is IgG. The present invention is also directed to a peptide derived from the monoclonal antibody highly specific for Bacillus spores. Preferably, the peptide can also bind the Bacillus spores specifically.
Also disclosed in the present invention is a method of preparing a monoclonal antibody highly specific for Bacillus spores, comprising the steps of immunizing an animal with the Bacillus spores and fusing local lymph nodes of the animal.
The present invention also is directed to a method of detecting anthrax in a field sample using a monoclonal antibody highly specific for Bacillus anthracis spores by contacting the sample with the monoclonal antibody and measuring the amount of binding of the antibody to the sample compared to an appropriate control.
The present invention also is directed to a method of detecting lethal Bacillus spores in a field sample using the peptide derived from the monoclonal antibody highly specific for Bacillus spores by contacting the sample with the peptide and measuring the amount of binding of the peptide to the sample compared to an appropriate control.
The following terms have the definitions set below.
As used herein, “hybridoma” refers to a continuously growing antibody-secreting cell line derived from the fusion of a specific normal antibody-forming B cell from an immunized mouse with an immortal myeloma cell line. Hybridomas secrete monoclonal antibodies described herein.
As used herein, “homogeneous staining” refers to the uniform staining at a similar intensity of all spores in a given sample.
As used herein, “heterogeneous staining” refers to the staining of one or more populations of spores in a given sample.
As used herein, “unfixed untreated spores” refers to spores that are in their native state in water and not treated with any kind of fixation reagent such as formalin or glutaraldehyde or paraformaldehyde.
As used herein, “two-color flow cytometric analysis” refers to the identification of spore subpopulations or other particles by fluorescence activated flow cytometry using two independent fluorochrome labeled antibodies.
As used herein, “limiting dilution” refers to the distributing of hybridoma cells into tissue culture plates such that less than 30% of the wells contain a growing clone. Each well, according to the Poisson distribution, should contain the progeny of only one cell.
The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.
Example 1
Animals
Eight to twelve-week-old BALB/c mice were purchased from Charles River Laboratories (Raleigh, N.C.) or bred in our mouse facility. BALB/c mice were used for immunization and obtaining feeder cells for subcloning of hybridomas, phenotypic analysis and functional studies. Mice were housed in accordance with institutional policies for animal care and usage.
Example 2
Bacterial Spores
B. subtilis spores were provided by Dr. Chuck Turnbough. B. anthracis spores were obtained from Dr. Joany J. Jackman at USAMIRID and B. thuringiensis spores were obtained from Abbott Laboratories.
Example 3
Antibody Production: Immunization and Fusion
Six-week-old female BALB/c mice were inoculated with 5×108 spores emulsified in complete Freund's adjuvant at day 0, and then repeatedly with spores in saline at days 3, 6, 9, 13, 17 and 20 in subcutaneous sites in the rear legs and inguinal regions.
On day 21, popliteal, inguinal and iliac lymph nodes draining sites of injection were removed, a lymphocyte suspension was prepared and fused to P3×63Ag8.653 using a modification of the method described by Kohler and Milstein. Fused cells were plated on ten 96-well plates in DMEM supplemented with 20% fetal bovine serum (FBS), 2 mmol/L L-glutamine, HAT medium, and FCS (from HyClone Laboratories Inc., Logan, Utah; other reagents from Sigma), and placed in a 37° C. incubator with 9% CO2.
Example 4
Primary Screening and Subcloning
Hybridoma supernatants were screened on spore suspensions using two-color flow cytometric analysis. Binding of secreted mouse Ig from supernatants to the spores was traced with Phycoerythrin (PE)-conjugated goat anti-mouse Ig (Southern Biotechnology Associates, Birmingham, Ala.). Data from stained cell samples were acquired using a FACScanTM or FACSCaliburTM flow cytometer with lysis II and Cell Quest software packages (Becton Dickinson, Mountain View, Calif.) and analyzed with WinList 2.01 (Verity Software House, Inc.) and WinMDI 2.0 software programs (Trotter@scripps.edu).
Example 5
ELISA
Flat bottom ELISA plates (E.I.A.A/2 plates, Costar) were coated with poly-L-Lysine (50 μg) for 30 minutes and a suspension of spores at 2×108/ml (40 μl) in distilled water were allowed to dry on the plates overnight. Supernatants were added and after incubation developed with goat anti-mouse Ig. Between each step, the plate was washed five times with PBS. The plate was developed with alkaline phosphatase substrate (Sigma, St. Louis, Mo.) (1 mg/ml) in substrate buffer (pH 9). For quantitative ELISA, mouse antibody of known concentration was used as a standard in each plate and OD405 values of plates were read by a Titertek Multiskan Plus MKIITM spectrophotometer (Flow, McLean, Va.). Antibody concentration was determined using an ELISALITETM program (Meddata, New York, N.Y.).
Example 6
Antibody Purification and Conjugation
Pure anti-spore antibodies were prepared from bulk hybridoma cultures by protein G chromatography. FITC an d phycoerythrin conjugates were prepared using standard procedures.
Example 7
Immunofluorescence and Immunohistochemical Analysis of Tissue Sections and Cytocentrifuge Preparations
Spleens embedded in OCTTM compound (Lab-Tek Products, Naperville, Ill.) were flash frozen in liquid nitrogen. Frozen sections were cut, air dried, fixed in ice-cold acetone, blocked with normal horse serum, and macrophages stained with MOMA-1 (rat, IgG2a, 10 μg/ml, from Dr. Georg Kraal), each developed with biotin-conjugated goat anti-rat IgG (SBA). Next, the sections were blocked with normal rat serum followed by anti-spore reagents and secondary reagents and streptavidin AMCA (Vector Laboratories, Burlingame, Calif.). Spore suspension in distilled water were dryed onto poly-L-Lysine treated glass slides for 2 hours at 37° C., blocked with 1% BSA and PBS and stained with antibodies for microscopy of spore suspensions.
Tissue sections and slides with dried spores were washed and mounted in Fluormount GTM (SBA, Birmingham, Ala.) and viewed with a Leica/Leitz DMRB fluorescence microscope equipped with appropriate filter cubes (Chromatechnology, Battleboro, Vt.). Images were acquired with a C5810 series digital color camera (Hamamatsu Photonic System, Bridgewater, N.J.) and processed with Adobe PhotoShopTM and IP LABTM Spectrum software (Signal Analytics Software, Vienna, Va.).
Example 8
DNA Sequencing Analysis
VH and VK gene sequencing was carried out from cDNA isolated from hybridomas. To make cDNA, total RNA was isolated from hybridomas using guanidinium thiocyanate-phenol-chloroform extraction. The cDNA was synthesized using an oligo-dT primer followed by PCR using a Cμ 3′ primer (SEQ ID NO: 1) and VH7183-specific primer (SEQ ID NO: 2) for the heavy chains or a CK 3′ primer (SEQ ID NO: 3) and a degenerate VK 5′ primer (SEQ ID NO: 4) for the light chains. The PCR amplified DNA was cloned into Bluescript II KSTM and subjected to sequencing using a SequenaseTM Kit (Strategene, La Jolla, Calif.). The DNA sequences were analyzed using the DNAstarTM program.
Example 9
Antibodies to B. subtilis 
Two immunization were made, one with fixed spores which gave only 3/192 (1.6%) monoclonal antibodies (mAbs) reactive with spores; and the other with unfixed spores which gave 95/384 (25%) (mAbs) reactive with spores, another 89 (20%) weakly reactive. These 576 clones were then tested against other spore components and 15 reacted with NAD synthetase, 6 with RNA polymerase, 5 with cot TC, 2 with SSPC, and 1 with cse60 by ELISA. Totally, 136 clones were reactive with spores or purified/recombinant components.
Among the clones reactive with the intact spores, certain patterns were observed: (1) two clones appeared to dramatically alter the FSC/SSC profile of spores on flow cytometry; (2) some clones reacted with germinated, but not with ungerminated spores; and (3) the majority had homogenous staining of germinated, but heterogeneous staining of ungerminated spores (FIG. 1).
96 clones of hybridomas reactive with B. subtilis were picked up and grown on a new plate. They include two negative clones, the clones reactive with purified proteins or peptides and clones reactive with spores representing different patterns. These antibodies were tested on the vegetative forms of B. subtilis (i.e., live bacteria) and were found to be negative (FIG. 2). They also did not react with two other species of spore-forming Bacilli (FIG. 3). Isotyping of the antibodies produced by these clones revealed that many (55/96) use λ light chains. Additionally, it was also unusual that 4 of these antibodies use α heavy chains.
All 96 clones were subcloned by limiting dilution and tested by flow cytometry. 68/96 were still reactive with spores and all except one were monoclonal. The reactive clones can be basically separated into two groups: those reactive with all spores and the other reactive with subsets of spores. Since these antibodies are of different isotypes, multiple parametric flow cytometric analysis could be done next. These important results showed that fixation of spores did not permit production of antibodies to the intact native spores and it was only when unfixed untreated spores were used to immunize mice could many highly specific antibodies to B. subtilis be isolated.
Example 10
Sequence Analysis of Monoclonal Antibody to B. subtilis 
The striking over usage of λ light chains in the antibodies led to sequencing the heavy and light chains of the genes from hybridomas synthesizing the B. subtilis spore specific antibodies to obtain an idea of the heterogeneity of antibodies generated. The heavy chains revealed remarkable homogeneity of VH gene usage in that all hybridomas used a member of VH7183 (MOPC21) family.
This member VH7183.6 appears to be the most unique member of this family in the framework three (FR3) region as shown in FIG. 4. The CDR3 region was diverse in nearly all cases and used variable DH and JH genes. These results suggest that there is a very strong selection for the use of this VH gene despite the similarities inherent in the family members of this family. λ light chain sequence showed the exclusive use of Vλ1 Cλ1 with different CDR3 regions. Six of these were selected for further study and characterization.
Example 11
Isolation of FR3 Peptides Which Bind to Spores
Based on the sequences of VH genes utilized in antibodies against Bacillus subtilis spores, two peptides were designed: one corresponding to the consensus sequence of these antibodies in the framework 3 region (Peptide Anti-spore: SEQ ID NO: 5), and the other corresponding to the consensus sequences of the 7183 VH gene family to which the particular VH gene belongs (Peptide 7183 consensus: SEQ ID NO: 6).
The carboxyl-terminal cysteine was added for fluorochrome conjugation. Both peptides were conjugated with phycoerythrin, and tested for their ability to bind Bacillus subtilis spores. 7183 consensus peptide was designed to be a control. It was found that the peptides derived from the anti-spore antibody stained brightly at 2 μg/ml (1 μM), while the consensus peptide stained spores at 200 μg/ml (100 μM) (FIG. 5). Thus the peptide derived from the spore specific nucleotide derived antibody sequence bind strongly and specifically to B. subtilis. 
Example 12
Serum Antibody Response to B. subtilis Spore Immunization
The immune response to Bacillus subtilis spores was characterized in mice. BALB/c mice were immunized with either spores or PBS (control). The mice were bled at 1, 2 and 3 weeks after immunization. Serum antibodies of different isotypes specific for spores were quantitated using ELISA. It was found that (1) immune responses peaked at 1 week; (2) light chain-containing antibodies account for about 30% of total spore-specific immunoglobulins; and (3) in contrast to all other isotypes, IgG3 antibodies continue to increase over the 3-week period. These findings confirmed the hybridoma analysis that the immune response to B. subtilis spores is dominated by a particular set of B cell clones.
Example 13
Monoclonal Antibodies to B. anthracis 
Mice were immunized with a 50:50 mix of heavily irradiated (4×106 Gy) Bacillus anthracis spores of the Ames and Sterne strains, generated hybridomas, and screened for antibody production by FACS analysis. About 60 hybridomas were selected for further characterization. A similar pattern of reactive antibodies was obtained with some of these panels binding 100% of B. anthracis spores. A seen in FIG. 6, a representative profile of more than 36 anti-anthrax antibodies which stain all spores but were not at all reactive with B. subtilis and B. thuringiensis spores. The VH gene sequences were determined and are shown in FIG. 7. Again a similar conservation in VH usage was found similar to what was found in antibodies to B. subtilis. In this case, one of the two VH genes is from VH7183 and the other from the VHJ558 family predominates. A third VH gene is from the VH Q52 family.
Example 14
Monoclonal Antibodies to B. thuringiensis 
A similar strategy was used to isolate and characterize  ̃100 antibody forming hybridomas which reacted with B. thuringiensis. Again the pattern was similar with all reacting with B. thuringiensis but not B. subtilis or B. anthracis. These antibodies were cloned and are sequenced. The discriminatory ability of antibodies is shown in FIGS. 8 and 9 where it is possible to clearly discriminate three distinct spore staining by fluorescence in a mixture of the three kinds of spores in vitro and in vivo.
Example 15
Discussion
The work presented here disclosed panels of antibodies which are highly specific and can discriminate between spores of the Bacillus family including the strategically important B. anthracis (anthrax). This is the first time such antibodies have been isolated and characterized. The reagents used in the various Divisions of the Armed services for testing were not monoclonal. They were made in sheep and other species against B. subtilis and B. anthracis. 
The antibodies disclosed in the present invention are unique because of several reasons: (1) the spores were not fixed with glutaraldehyde or formalin (which chemically modifies the spores) before immunization; (2) these are monoclonal antibodies made by immunizing and fusing local lymph nodes. Such a procedure has not been used in the past. The few monoclonal antibodies described before have been of the IgM isotypes which are more difficult antibodies to use and are more cross-reactive, i.e., react with spores other than B. anthracis, while the monoclonal antibodies disclosed herein are IgG. IgM antibodies of this kind are useless in instruments designed to give positive results for anthrax spores in the field, since such antibodies will also detect harmless spores such as B. subtilis which is ubiquitous in the environment.
The amino acid sequences of these anti-spore antibodies were also analyzed, which allows one to design and make smaller peptide molecules which can also bind spores. These will be more rugged molecules than the large antibody molecule and can be used in other kinds of detectors. Such peptides are totally unique in their binding to Bacillus spores.
There are numerous government (services and intelligence), as well as private groups trying to make instruments that are small, portable and highly accurate in their detection of small numbers of potentially lethal spores such as anthrax. The monoclonal antibodies presented here could play a critical role in their instrument development program. Such findings will be significant in detecting air and water containing anthrax spores for civilian and military use.
Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. These patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.
One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present examples along with the methods, procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.

## Claims

1. A method of preparing a monoclonal IgG antibody specific for Bacillus anthracis spores, comprising the steps of:
immunizing an animal with unfixed Bacillus anthracis spores; and
fusing lymph nodes located at the draining site of the immunization of said animal with a myeloma cell to produce a hybridoma cell that produces a monoclonal antibody specific for Bacillus anthracis spores.

2. The method of claim 1, wherein said animal is a mouse.

3. A method of detecting Bacillus anthracis spores in a field sample using a monoclonal IgG antibody specific for said spores, comprising the steps of:
contacting said sample with a monoclonal antibody specific for Bacillus anthracis spores, wherein the monoclonal antibody is prepared by a method comprising immunizing an animal with unfixed Bacillus anthracis spores; and
detecting the presence of the monoclonal antibody, wherein the presence of the monoclonal antibody indicates the presence of spores.

4. The method of claim 1, wherein the unfixed spore is irradiated prior to immunizing the animal.

5. A method of detecting Bacillus anthracis spores in a field sample using a monoclonal antibody specific for said spores, comprising the step of:
contacting said sample with a monoclonal antibody prepared by the method of claim 1; and
detecting the presence of the monoclonal antibody, wherein the presence of the monoclonal antibody indicates the presence of spores.