Patent Application
20090297548
Bacillus anthracis is a facultative anaerobic, non-motile, gram positive, endospore-forming bacillus, which primarily causes a fatal disease in herbivores (Mock, M. and A. Fouet. 2001. Anthrax. Annu. Rev. Microbiol. 55:647-671). Human infection is acquired upon exposure to endospores and, depending on the route of infection, the disease may manifest as cutaneous (least dangerous and easily treatable), inhalational (often fatal) or gastrointestinal anthrax (rare) (Leppla, S. H., et al., 2002. J. Clin. Invest. 110:141-144; Mock, M. and A. Fouet. 2001). Irrespective of the route of infection, progression to systemic disease can occur. Endospores phagocytosed by macrophages are transported to the regional lymph nodes where they germinate into vegetative bacilli (Leppla, S. H., et al., 2002; Mock, M. and A. Fouet. 2001), which then multiply in the lymphatic system and disseminate into the blood stream causing massive septicemia. The organism then elaborates virulence factors that cause a variety of systemic effects leading to death of the host (Leppla, S. H., et al., 2002; Mock, M. and A. Fouet. 2001).
Thus far, the pathogenicity of B. anthracis has been attributed to the production of virulence factors encoded on two virulence plasmids, pXO1 and pXO2, which are present in all fully virulent strains. pXO2 encodes an antiphagocytic, γ-D-glutamic acid capsule. pXO1 encodes three virulence proteins, protective antigen (PA), lethal factor (LF) and the edema factor (EF), which assemble to form two binary toxins. PA, the non-toxic, receptor-binding moiety can assemble with either EF to form edema toxin (ET), or with LF to form lethal toxin (LT). The enzymatic moiety of ET is an adenylate cyclase (Mock, M. and A. Fouet. 2001) that acts by increasing intracellular levels of cAMP, which is responsible for the edema typical in patients with cutaneous anthrax. The enzymatic moiety of LT is a zinc metalloprotease (Mock, M. and A. Fouet. 2001) that exerts its effect by cleaving mitogen-activated protein kinase kinase (MAPKK). The precise mechanism by which LT causes death in systemic anthrax is still under investigation. Results of recent studies in mice implicate hypoxia-induced tissue injury (Moayeri, M., et al., 2003. J. Clin. Invest. 112:670-682) and genetic factors (Moayeri, M., et al., 2004. Infect. Immun. 72:4439-4447) in LT-mediated lethality, rather than induction of proinflammatory cytokines, as suggested earlier (Mock, M. and A. Fouet. 2001). In addition to the above, results of several recent studies have alluded to other unidentified virulence determinants acting in concert with the aforementioned factors to play a contributory role in anthrax pathogenesis (Brossier, F., et al. 2002. Infect. Immun. 70:661-664; Cohen, S., et al., 2000. Infect. Immun. 68:4549-4558; Little, S. F. and G. B. Knudson, 1986. Infect. Immun. 52:509-512; Pezard, C., M. et al., 1995. Infect. Immun. 63:1369-1372; Stepanov, A. V., et al., 1996. J. Biotechnol 44:155-160; Welkos, S., et al., 2001. Microbiology 147:1677-1685).
Although therapeutic options are available to successfully treat the syndromes of anthrax upon early diagnosis, vaccination may be the most effective strategy to thwart the disease (Leppla, S. H., et al., 2002), especially in target populations likely to be exposed to anthrax spores, such as military personnel and workers in wool and leather industries. Vaccination also remains the most economical means of mass immunization. The anthrax vaccine currently approved for human use in the United States, Anthrax Vaccine Adsorbed (AVA), is a cell-free filtrate prepared from formalin-treated, culture supernatant of a non-proteolytic, toxigenic and unencapsulated, avirulent B. anthracis strain (pXO1+, pXO2−), V770-NP1-R, adsorbed to the adjuvant, aluminum hydroxide (Joellenbeck, L. M., et al., 2002. National Academy Press, Washington, D.C.). It is administered subcutaneously in a volume of 0.5 ml at 0, 2, and 4 weeks and at 6, 12 and 18 months. Thereafter, boosters administered annually are essential to maintain protective immunity (Friedlander, A. M., et al., 1999. JAMA 282:2104-2106; Leppla, S. H., et al., 2002). A similar vaccine, prepared by adsorbing a sterile culture supernatant-filtrate of the 32F2 Sterne strain to potassium aluminum sulfate is licensed for use in the United Kingdom (Leppla, S. H., et al., 2002; Whiting, G. C., et al., 2004. Vaccine 22:4245-4251). Studies have demonstrated that AVA is safe (Joellenbeck, L. M., et al., 2002) and protects against both cutaneous (Joellenbeck, L. M., et al., 2002; Leppla, S. H., et al., 2002) and inhalational anthrax (Friedlander, A. M., et al., 1999. JAMA 282:2104-2106; Joellenbeck, L. M., et al, 2002; Leppla, S. H., et al., 2002).
Despite documentation attesting to safety and efficacy of AVA, currently approved human-use anthrax vaccines have several limitations. Immunization with human-use acellular, PA-based vaccines reportedly induces low and transient immune responses (Hambleton, P., et al., 1984. Vaccine 2:125-132; Lincoln, R. and D C Fish. 1970. Anthrax toxin, p. 361-414; T. C. Monte, et al., Academic Press, Inc., New York), and, consistent with this observation, multiple administrations of AVA are required for induction of protective immunity (Brachman, P. S., et al., 1962. Am. J. Public Health 52:632-645). Immunization is associated with local and sometimes systemic reactogenicity attributable to residual LF and EF, which may combine with PA to form active LT and ET, the adjuvant used, and also to the presence of uncharacterized components in vaccine preparations (Joellenbeck, L. M., et al., 2002; Turnbull, P. C. 1991. Vaccine 9:533-539; Whiting, G. C., et al., 2004. Vaccine 22:4245-4251). An additional limitation of AVA includes the lack of standardization in the manufacturing process resulting in batch to batch variations in the amount of PA and the unavailability of reliable assays to measure potency of vaccine preparations (Leppla, S. H., et al., 2002).
Due to the limitations of currently known B. anthracis vaccines, including AVA, there is a need for development of a defined anthrax vaccine free of significant adverse effects and capable of inducing sustained protective immunity.
In one embodiment, the invention provides an immunogenic composition comprising at least one anthrax spore-associated protein or immunogenic fragment and/or functional variant thereof.
In another embodiment, the invention provides an immunogenic composition comprising at least one expression vector, wherein the expression vector comprises a nucleic acid molecule encoding an anthrax spore-associated protein or immunogenic fragment and/or functional variant thereof. The expression vector may comprise at least one additional nucleic acid molecule encoding an anthrax spore-associated protein or immunogenic fragment and/or functional variant thereof. Furthermore, the expression vector may be a viral vector or a plasmid vector.
In one embodiment, the immunogenic composition of the invention further comprises protective antigen (PA) (or immunogenic fragment and/or functional variant thereof) or a nucleic acid molecule encoding the PA or a immunogenic fragment and/or functional variant thereof.
In one embodiment, the immunogenic composition of the invention is acellular.
In another embodiment, the immunogenic composition of the invention induces an immunological response in a subject against Bacillus anthracis. The immunological response induced in the subject may be against Bacillus anthracis in the spore form and/or in the bacillus form. The subject may be a mammal. The mammal may be a human.
In one embodiment, the immunogenic composition of the invention further comprises a pharmaceutically acceptable excipient. In another embodiment, the immunogenic composition of the invention further comprises an adjuvant.
In one embodiment, the invention provides an immunogenic composition comprising at least one anthrax spore-associated protein having an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, and immunogenic fragments and/or functional variants thereof.
In another embodiment, the invention provides an immunogenic composition comprising at least one expression vector, wherein the expression vector contains a nucleic acid molecule encoding an anthrax spore-associated protein or immunogenic fragment and/or functional variant thereof, having a nucleic acid sequence is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, and fragments thereof.
In one embodiment, the invention provides a method for inducing an immunological response in a subject comprising administering to said subject an immunogenic composition comprising at least one anthrax spore-associated protein (or immunogenic fragment and/or functional variant thereof) or an immunogenic composition comprising at least one expression vector, wherein the expression vector comprises a nucleic acid molecule encoding an anthrax spore-associated protein or immunogenic fragment and/or functional variant thereof. The subject may be uninfected with Bacillus anthracis. The subject may be a mammal. The mammal may be a human.
In another embodiment of the method of the invention, the immunogenic composition comprises PA (or an immunogenic fragment and/or functional variant thereof) or a nucleic acid molecule encoding PA (or an immunogenic fragment and/or functional variant thereof). In one embodiment, the subject is uninfected with Bacillus anthracis. In another embodiment, the subject is infected with Bacillus anthracis. In one embodiment of the method of the invention, the administering occurs about one to about sixty days after infection, when the Bacillus anthracis spores have not yet germinated. If the spores have germinated, the administering may be effected in concert with an additional therapy against Bacillus anthracis infection. In one embodiment, the additional therapy comprises antibiotic therapy.
In one embodiment of the method of the invention, the immunological response is against Bacillus anthracis. Bacillus anthracis may exist in the spore form (i.e., in the form of a spore formed by the bacteria) and/or in the bacillus form (i.e., upon activation (germination) of the spore; in this form, the bacteria can reproduce). Accordingly, the immunological response may be against Bacillus anthracis in the spore form and/or the bacillus form.
In another embodiment of the method of the invention, the amount of immunological response is effective to confer substantial protective immunity against infection with Bacillus anthracis in the subject.
In yet another embodiment of the method of the invention, the immunogenic composition is administered 1 to 2 times.
Methods of the invention can further comprise the step of obtaining the anthrax spore-associated protein (or an immunogenic fragment and/or functional variant thereof).
In one embodiment, the invention provides a kit comprising an immunogenic composition comprising at least one anthrax spore-associated protein (or an immunogenic fragment and/or functional variant thereof) or an immunogenic composition comprising at least one expression vector, wherein the expression vector comprises a nucleic acid molecule encoding an anthrax spore-associated protein or immunogenic fragment and/or functional variant thereof and optionally instructions for administering the immunogenic composition to induce an immunological response in a subject and optionally a device and/or vessel for the administration of the composition.
Other aspects of the invention are described in or are obvious from the following disclosure, and are within the ambit of the invention.
The following Detailed Description, given by way of example, but not intended to limit the invention to specific embodiments described, may be understood in conjunction with the accompanying drawings, in which:
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide one of skill in the art to which this invention pertains with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2d ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); Hale & Marham, The Harper Collins Dictionary of Biology (1991); and Lackie et al., The Dictionary of Cell & Molecular Biology (3d ed. 1999); and Cellular and Molecular Immunology, Eds. Abbas, Lichtman and Pober, 2nd Edition, W.B. Saunders Company. For the purposes of the present invention, the following terms are further defined.
The term “anthrax vaccine” refers to a vaccine administered in any known form, such as, for example, a protein antigen, such as a spore protein, or a nucleic acid encoding the spore protein, or some combination thereof, that is specifically immunoreactive against Bacillus anthracis, the causative agent of anthrax, wherein an immune response is generated against the vaccine which in turn immunizes the subject against infection by B. anthracis. Alternatively, or at the same time, the anthrax vaccine can also refer to a vaccine composition that elicits an immune response against anthrax toxins, such as, for example, protective antigen.
The term “anthrax spore-associated protein” refers to any protein obtained or derived from the spore (e.g., interior or exterior) or spore form of a Bacillus anthracis isolate strain or the like.
The phrase “specifically immunoreactive” can refer to a binding reaction between an antibody and a protein, compound, or antigen, having an epitope recognized by the antigen binding site of the antibody. This binding reaction is determinative of the presence of a protein, antigen or epitope having the recognized epitope amongst the presence of a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies can bind to a protein having the recognized epitope and bind, if at all, to a detectably lesser degree to other proteins lacking the epitope which are present in the sample. An antibody that is specifically immunoreactive with an antigen can bind to that antigen and form a complex therewith. In an in vivo context, “specifically immunoreactive” can refer to the conditions under which in an animal forms an immune response against a vaccine or antigen, e.g. a humoral response to the antigen (the production of antibodies, against a vaccine, protein, compound, or antigen presented thereto under immunologically reactive conditions) or a cell-mediated (also herein as “cellular immune response”, i.e. a response mediated by T lymphocytes against the vaccine, protein, compound or antigen presented thereto).
The term “immunity” can refer to both “natural” (native or innate) immunity or “acquired” (specific) immunity. Natural immunity relates to a collection of innate mechanisms in a subject that are capable of warding off or protecting against infection by a foreign organism, virus or substance, such as, physical barriers, phagocytic cells and eosinophils in the blood and tissues, natural killer cells, and various blood-borne molecules (e.g. complement system) that are already present in a subject prior to infection by the invading foreign organism, virus or substance. Acquired or specific immunity refers to immunity to a foreign organism, virus or substance (i.e. the antigen) that is induced by the presence of the invading organism, virus, or substance which encompasses both humoral and cell-mediated mechanisms.
An “immunogenic composition” is an antigenic preparation of the invention, including, e.g., a protein or immunogenic fragment thereof or a polynucleotide encoding a protein or immunogenic fragment thereof or a polysaccharide, a combination of more than one protein or immunogenic fragment thereof, or a combination of a protein (or immunogenic fragment thereof) and a polynucleotide encoding a protein (or immunogenic fragment thereof) administered to stimulate the recipient's humoral and cellular immune systems to one or more of the antigens present in the vaccine preparation. The term “immunogenic composition” includes the terms vaccine and immunological composition. “Vaccination” or “immunization” is the process of administering an immunogenic composition and stimulating an immune response to an antigen.
An “antigen” or “immunogen” is any agent, e.g., a polynucleotide, a protein, a peptide, or a polysaccharide, that elicits an immune response and is therefore characterized as “immunogenic.” The antigen can be attached to an invading organism or virus, e.g. a cell surface protein or viral capsule protein, or unattached, e.g. a circulating anthrax toxin.
An “immune response” refers to the activities of the immune system in response to an invading antigen, organism, virus, or substance, including mechanisms relating to natural and acquired immunity, and humoral and cell-mediated immunity, including especially the induction of antigen-specific antibodies and the activation and proliferation of specific cytotoxic T-cells after contact with an antigen, organism, virus or substance.
The term “antibody” refers to the family of glycoproteins encoded by an immunoglobulin gene(s) produced in connection with a humoral immune response which specifically recognize and bind to antigens to which they are raised. In the body, antibodies can be produced in a membrane-bound form by B lymphocytes as well as in a secreted form by progeny of B cells that differentiate in response to antigenic stimulation. The term “antibody” can further refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE. The term “antibody” is also used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab′, Fab, F(ab′)2, single domain antibodies (DABs), Fv, scFv (single chain Fv), and engineering multivalent antibody fragments such as dibodies, tribodies and multibodies. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art. Means for preparing and characterizing antibodies are also well known in the art (See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference).
The anthrax “protective antigen” (PA) is an 83 kDa protein (SEQ ID NO:159) produced by Bacillus anthracis. PA is one of two protein components of the lethal or anthrax toxin produced by B. anthracis. The 83 kDa PA binds at its carboxyl-terminus to a cell surface receptor, where it is specifically cleaved by a protease, e.g., furin, clostripain, or trypsin. This enzymatic cleavage releases a 20 kDa amino-terminal PA fragment, while a 63 kDa carboxyl-terminal PA fragment remains bound to the cell surface receptor. The description of protective antigen includes binary toxin functional equivalents such as protein Ib of C. perfringens.
“Parenteral” administration of a vaccine includes, e.g., subcutaneous, intravenous, intramuscular, or intrasternal injection or infusion techniques.
“Antigen presenting cells” are cells, e.g., dendritic cells or macrophages, that process peptide antigens through the MHC class I processing pathway so that the antigen-MHC class I complex is displayed on their cell surface. A “dendritic” cell is a motile, non-phagocytic adherent cell that acts as an efficient antigen-presenting cell and moves readily between the lymph nodes and other organs. Dendritic cells are further classified into subgroups, including, e.g., follicular dendritic cells, Lagerhans dendritic cells, and epidermal dendritic cells.
“Anthrax toxin” is a binary toxin produced by B. anthracis, composed of LF and PA. Anthrax toxin may also refer to the binary edema toxin of B. anthracis, composed of LF and EF (edema factor). A “binary toxin” is a bacterial toxin that is composed of two separate proteins that associate to form the toxin.
“Substantial protective immunity” refers to a state in which the subject's body responds specifically to the antigen(s), and a protective response is mounted against the pathogenic agent (in this case, Bacillus anthracis), said response comprising an alteration in the reactivity of the subject's immune system in response to the antigen(s), potentially involving antibody production, induction of cell-mediated immunity, and/or complement activation. The response results in a degree of protection (i.e., a protective immune response) comprising protection from Bacillus anthracis infection, or further infection or spread of infection if the subject is already infected with Bacillus anthracis.
An “expression vector” is a vector used for transfer of genetic information (in the form of a nucleotide sequence) into a cell, where a recombinant protein encoded by said genetic information can then be expressed.
The term “obtaining” as in “obtaining the spore associated protein” is intended to include purchasing, synthesizing or otherwise acquiring the spore associated protein (or indicated substance or material).
It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.
Peptide-Based Immunogenic Compositions
In one aspect, the present invention is directed to immunogenic compositions comprising at least one antigen that is capable of eliciting an immune response and of providing a protective effect against B. anthracis or a toxin thereof.
One embodiment provides an immunogenic composition of the invention that comprises at least one anthrax spore-associated protein or a variant form thereof or an immunogenic fragment thereof. As used herein, the term “immunogenic fragment thereof” can refer to a peptide which is at least 6 amino acids in length, preferably at least about 15 amino acids in length, and has the ability to elicit production of antibodies that bind to the wild-type protein from which it is derived, and the ability to elicit an immune response and protective effect that is the same or substantially the same as the immune response and protective effect elicited by the native protein from which it is derived.
It will be appreciated by the person of skill in the art to which the present invention pertains that there are numerous possible ways to determine whether a particular antigen fragment of the invention is an “immunogenic fragment” of the antigens of the invention (e.g. anthrax spore-associated proteins or anthrax PA). The invention encompasses any method for measuring, evaluating or determining whether an antigen fragment is immunogenic, including, for example, in vitro or in vivo testing. For example, in in vitro methods, an immunogenic antigen fragment of interest can be tested using antibody-binding assays, e.g. immunoassays, that compare the strength of antibody binding to the native antigen and the immunogenic antigen fragment of interest. A detailed review of immunological assay design, theory and protocols can be found in numerous texts in the art, including “Practical Immunology”, Butt, W. R., ed., (1984) Marcel Dekker, New York and “Antibodies, A Laboratory Approach”, Harlow et al. eds. (1988) Cold Spring Harbor Laboratory. In in vivo methods, an immunogenic antigen fragment of interest can be tested in an animal, such as a mouse or rabbit or cow, to determine if the animal produces antibodies raised against the antigen fragment of interest that are capable eliciting or establishing a protective response or alternatively, if the antibodies formed against the immunogenic antigen fragment of interest specifically react with the native antigen from which the antigen fragment is derived.
Antigen fragments that are similarly immunogenic or substantially immunogenic as the native antigens of the invention, e.g. the anthrax spore-associated proteins of the invention or anthrax PA, can be prepared in any suitable manner available to one of ordinary skill in the art. Such methods can include genetic engineering methods, whereby a nucleic acid molecule encoding only a partial amino acid sequence (i.e. antigen fragment) of the native antigen is prepared and used to either express the antigen fragment or is used to administer to a subject for achieving in vivo expression of the antigen fragment. Physical and/or chemical and/or enzymatic methods can also be used to prepare the immunogenic fragments of the invention, including, for example, peptidase treatment or chemical cleavage. Methods for producing immunogenic fragments of the inventive anthrax spore-associated proteins and PA by way of physical and/or chemical and/or enzymatic methods can be found in the technical literature, for example, in Methods in Enzymology, Volume 182, Guide to Protein Purification, Eds. J. Abelson, M. Simon, Academic Press, 1st Edition, 1990. In addition, immunogenic antigen fragments of the invention can be synthesized using known and available methods and techniques for protein/peptide synthesis, for example, as described in Chemical Approaches to the Synthesis of Peptides and Proteins (Hardcover), Eds. P. Lloyd-Williams, F. Albericio, and E. Giralt, CRC Press, 1st Edition, 1997.
In certain embodiments, the anthrax spore-associated protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, and immunogenic fragments or functional variants thereof.
The anthrax spore-associated proteins of the present invention can be a full-length, wild-type, mature anthrax spore-associated protein, i.e. “native protein.” The term “anthrax spore-associated protein”, as used herein, also can encompass naturally-occurring and man-made variant anthrax spore-associated proteins whose amino acid and/or nucleotide sequences differ from the sequences shown herein. Such variant proteins can have an amino acid sequence which is at least 90% identical, preferably at least 95% identical, or more preferably at least 99% identical to the specific amino acid sequences shown herein. Such variant proteins can have an altered sequence in which one or more of the amino acids in the specific anthrax spore-associated protein sequence is substituted, or in which one or more amino acids are deleted from or added to such sequence. Such variants include degenerate variants. Such variants, when injected into an animal, elicit production of antibodies that bind to the mature, wild-type anthrax spore-associated protein in question, i.e., the anthrax spore-associated protein whose sequence corresponds to one of those depicted herein.
The term “variant form thereof,” or equivalently “functional variant thereof” as used herein, can refer to a distinct but related version of the at least one anthrax spore-associated protein or other proteins of the invention (e.g. the B. anthracis PA) that can differ with respect to the amino acid sequence of the variant as compared to the native protein, the underlying nucleotide sequence encoding the variant as compared to the native nucleotide sequence, or the state of chemical modification of the variant as compared to the native protein, e.g. glycosylation pattern. The functional variant forms of the antigens of the invention include both those that are created by man, e.g. chemical modification or genetic engineering, or those that are produced in nature, e.g. by naturally occurring genetic mutation. The functional variants of the invention can differ from the native antigens as a result of conservative/degenerate nucleotide and/or amino acid sequence substitutions. Preferably, the functional variants of the invention will contain at least 90% sequence identity, more preferably at least 95% sequence identity, and still more preferably, at least 99% sequence identity with the native proteins of the invention, e.g. the anthrax spore-associated proteins and/or the anthrax PA. Functional variants of the invention are functionally equivalent to the individual native antigens from which they derive or are otherwise obtained.
As used herein, the terms percent (%) sequence identity or percent (%) homology are used synonymously as a measure of the similarity of two or more amino acid sequences. Methods for determining percent (%) sequence identity or percent (%) homology are well known in the art.
For the purposes of the present invention, percent (%) sequence identity or homology can be determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity may be determined using any of a number of mathematical algorithms. A nonlimiting example of a mathematical algorithm used for comparison of two sequences is the algorithm of Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1990; 87: 2264-2268, modified as in Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1993; 90: 5873-5877.
Another example of a mathematical algorithm used for comparison of sequences is the algorithm of Myers & Miller, CABIOS 1988; 4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Yet another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm as described in Pearson & Lipman, Proc. Natl. Acad. Sci. USA 1988; 85: 2444-2448. Advantageous for use according to the present invention is the WU-BLAST (Washington University BLAST) version 2.0 software. This program is based on WU-BLAST version 1.4, which in turn is based on the public domain NCBI-BLAST version 1.4 (Altschul & Gish, 1996, Local alignment statistics, Doolittle ed., Methods in Enzymology 266: 460-480; Altschul et al., Journal of Molecular Biology 1990; 215: 403-410; Gish & States, 1993; Nature Genetics 3: 266-272; Karlin & Altschul, 1993; Proc. Natl. Acad. Sci. USA 90: 5873-5877; all of which are incorporated by reference herein).
In general, comparison of amino acid sequences is accomplished by aligning an amino acid sequence of a polypeptide of a known structure with the amino acid sequence of a the polypeptide of unknown structure. Amino acids in the sequences are then compared and groups of amino acids that are homologous are grouped together. This method detects conserved regions of the polypeptides and accounts for amino acid insertions and deletions. Homology between amino acid sequences can be determined by using commercially available algorithms (see also the description of homology above). In addition to those otherwise mentioned herein, mention is made too of the programs BLAST, gapped BLAST, BLASTN, BLASTP, and PSI-BLAST, provided by the National Center for Biotechnology Information. These programs are widely used in the art for this purpose and can align homologous regions of two amino acid sequences.
In all search programs in the suite the gapped alignment routines are integral to the database search itself. Gapping can be turned off if desired. The default penalty (Q) for a gap of length one is Q=9 for proteins and BLASTP, and Q=10 for BLASTN, but may be changed to any integer. The default per-residue penalty for extending a gap (R) is R=2 for proteins and BLASTP, and R=10 for BLASTN, but may be changed to any integer. Any combination of values for Q and R can be used in order to align sequences so as to maximize overlap and identity while minimizing sequence gaps. The default amino acid comparison matrix is BLOSUM62, but other amino acid comparison matrices such as PAM can be utilized.
Alternatively or additionally, the term “homology” or “identity”, for instance, with respect to a nucleotide or amino acid sequence, can indicate a quantitative measure of homology between two sequences. The percent sequence homology can be calculated as (Nref−Ndif)*100/−Nref, wherein Ndif is the total number of non-identical residues in the two sequences when aligned and wherein Nref is the number of residues in one of the sequences. Hence, the DNA sequence AGTCAGTC will have a sequence identity of 75% with the sequence AATCAATC (Nref=8; Ndif=2).
Alternatively or additionally, “homology” or “identity” with respect to sequences can refer to the number of positions with identical nucleotides or amino acids divided by the number of nucleotides or amino acids in the shorter of the two sequences wherein alignment of the two sequences can be determined in accordance with the Wilbur and Lipman algorithm (Wilbur & Lipman, Proc Natl Acad Sci USA 1983; 80:726, incorporated herein by reference), for instance, using a window size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4, and computer-assisted analysis and interpretation of the sequence data including alignment can be conveniently performed using commercially available programs (e.g., Intelligenetics™ Suite, Intelligenetics Inc. CA). When RNA sequences are said to be similar, or have a degree of sequence identity or homology with DNA sequences, thymidine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence. Thus, RNA sequences are within the scope of the invention and can be derived from DNA sequences, by thymidine (T) in the DNA sequence being-considered equal to uracil (U) in RNA sequences.
And, without undue experimentation, the skilled artisan can consult with many other programs or references for determining percent homology.
In one embodiment of the invention, the substitutions of the functional variants of the inventive antigens are conservative amino acid substitutions, in which the substituted amino acid has similar structural or chemical properties with the corresponding amino acid in the reference sequence. By way of example, conservative amino acid substitutions involve substitution of one aliphatic or hydrophobic amino acid, e.g. alanine, valine, leucine and isoleucine, with another; substitution of one hydroxyl-containing amino acid, e.g. serine and threonine, with another; substitution of one acidic residue, e.g. glutamic acid or aspartic acid, with another; replacement of one amide-containing residue, e.g. asparagine and glutamine, with another; replacement of one aromatic residue, e.g. phenylalanine and tyrosine, with another; replacement of one basic residue, e.g. lysine, arginine and histidine, with another; and replacement of one small amino acid, e.g., alanine, serine, threonine, methionine, and glycine, with another.
By way of example, functional variant sequences, which are at least 90% identical, have no more than 1 alteration, i.e., any combination of deletions, additions or substitutions, per 10 amino acids of the flanking amino acid sequence. Percent identity is determined by comparing the amino acid sequence of the variant with the reference sequence using MEGALIGN module in the DNA STAR program.
The term “anthrax spore-associated protein”, as used herein, can sometimes encompass functional variants and immunogenic antigen fragments that are encoded by polynucleotide variants, which are polynucleotides that differ from a reference polynucleotide. Generally, the differences are limited so that the nucleotide sequences of the reference and the variant are closely similar overall and, in many regions, identical. The present invention encompasses both allelic variants and degenerate variants.
As iterated briefly above, a variant of a polynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally. By an “allelic variant” is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism (Lewin, (1989), PNAS 86:9832-8935). Diploid organisms may be homozygous or heterozygous for an allelic form. Non-naturally occurring variants of the polynucleotide may be made by art-known mutagenesis techniques, including those applied to polynucleotides, cells or organisms.
Polynucleotide variants referred to as “degenerate variants” constitute polynucleotides which comprise a sequence substantially different from those described herein but which, due to the degeneracy of the genetic code, still encode a polypeptide comprised in an immunogenic composition of the present invention. That is, all possible polynucleotide sequences that encode the polypeptides defined herein as potentially comprised in an immunogenic composition of the present invention are contemplated. This includes the genetic code and species-specific codon preferences known in the art.
Nucleotide changes present in a variant polynucleotide may be silent, which means that they do not alter the amino acids encoded by the polynucleotide. However, nucleotide changes may also result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. The substitutions, deletions or additions may involve one or more nucleotides. The variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. In one embodiment of the present invention, the polynucleotide variants encode polypeptides which retain substantially the same biological properties or activities as the proteins identified herein.
In another embodiment, the peptide-based immunogenic composition of the invention comprises an anthrax spore-associated protein or an immunogenic fragment thereof and the B. anthracis PA protein or an immunogenic fragment thereof. The full-length, wild-type PA protein has a molecular weight of 83 kDA and comprises 735 amino acids. The full-length, wild-type, mature PA protein comprises the amino acid sequence, SEQ ID NO:160, shown herein. The term “PA protein”, as used herein, can also encompass wild-type and mutated PA proteins whose sequence differs slightly from the afore-mentioned sequence. Such variants have an amino acid sequence which is at least 90% identical, preferably at least 95% identical, more preferably at least 99% identical to the amino acid sequence in question. Suitable variants elicit production of antibodies that bind to the wild-type PA protein.
In one embodiment, the anthrax spore-associated protein and optional PA components of the immunogenic compositions of the invention are pure, meaning that the polypeptides have been isolated and purified to substantial homogeneity. A polypeptide that produces a single peak that is at least 95% of the input material on an HPLC column is considered “pure” for the purposes of this invention. Utilizing proteins of high purity may signify the absence of adjuvant materials such as alum, as well as the elimination of common contaminants or additives used in prior art anthrax vaccines.
Any known method of purification that is suitable for producing pure anthrax spore-associated protein or PA polypeptides or the immunogenic and/or functional variants thereof, may be used, for example, using chromatography, and can be found described in the technical literature, for example, in Methods in Enzymology, Volume 182, Guide to Protein Purification, Eds. J. Abelson, M. Simon, Academic Press, 1st Edition, 1990. Thus, suitable materials for performing such purification steps, such as chromatographic steps, are known to those skilled in the art.
In one embodiment of the present invention, the peptide-based immunogenic composition of the invention can be delivered to a subject in need thereof employing an attenuated bacterial vaccine vector, such as that described in U.S. Pat. No. 6,383,496, which is incorporated herein in its entirety by reference. Such vectors include, without limitation, attenuated strains of Vibrio cholerae, Salmonella typhimurium, Listeria monocytogenes, and lactococcal species. Attenuated bacterial vaccine vectors, such as those above, can effectively deliver proteins to the mucosal immune system, consequently engendering a protective mucosal immune response in the subject. Such vaccines and “carrier microbes” can serve as vehicles for delivering desired gene products such as the antigens of the invention, the immunogenic fragments thereof and functional variants thereof also of the invention, to subjects, including humans, as well as for delivering nucleic acids, either DNA or RNA, to target cells, such as human cells.
The attenuated microbes, i.e. attenuated bacterial vaccine vectors of the present invention, contain at least one recombinant gene capable of expressing a desired gene product, e.g. the antigens of the invention (and immunogenic fragments and functional variants thereof), which allows their use as carriers or delivery vehicles of the gene product to subjects, including humans. By delivery of the desired gene product it is meant that either the gene product or the polynucleotide, i.e. nucleic acid, either DNA or RNA, encoding the product is delivered to the subject.
Another aspect of the invention is directed to an immunogenic composition comprising at least one expression vector comprising a nucleic acid molecule that encodes an antigen of the invention, e.g. an anthrax spore-associated protein, or immunogenic fragment thereof, or functional variant thereof, which are capable of eliciting an immune response and a protective effect against B. anthracis or toxins thereof.
It is generally known that the mammalian system reacts to invading pathogens by mounting two broad defenses: the cell-mediated response and the humoral response. Viral and other intracellular infections are controlled primarily by the cell-mediated immune system. This control is achieved through recognition of foreign antigen displayed on the cell surface of an infected cell.
The cell-mediated immune system responds to endogenous antigen presented by the MHC class I processing pathway. Without being bound by theory, an objective for a vaccine that stimulates the cell-mediated immune system is to deliver protein antigen to the cell cytosol for processing and subsequent presentation by MHC class I molecules.
The use of deoxyribonucleic acid (DNA) molecules for vaccination has been known since the beginning of the 1990s (e.g. Wolf et al. Science 1990. 247. 1465-1468). This vaccination technique induces cellular and humoral immunity after in vivo transfection of cells of the subject to be vaccinated with DNA or RNA molecules encoding immunologically active proteins.
It will be appreciated that the use of DNA molecules for vaccination contrasts with “traditional” vaccination techniques which involve the introduction into an animal system of an antigen which can induce an immune response in the animal, and thereby protect the animal against infection. Following the observation in the early 1990's that plasmid DNA could directly transfect animal cells in vivo, significant research efforts have been undertaken to develop vaccination techniques based upon the use of DNA plasmids (and other deliverable forms of DNA molecules) to induce immune responses, by direct introduction into animals DNA which encodes for antigenic peptides. Such techniques, which are referred to as “DNA immunization” or “DNA vaccination” have now been used to elicit protective antibody (humoral) and cell-mediated (cellular) immune responses in a wide variety of pre-clinical models for viral, bacterial and parasitic diseases. Such techniques are contemplated by the present invention.
DNA vaccines can consist of a bacterial plasmid vector into which is inserted a viral promoter, a gene of interest which encodes for an antigenic peptide and a polyadenylation/transcriptional termination sequence. The gene of interest may encode a full protein (e.g. anthrax spore-associated protein of the invention) or simply an antigenic peptide (e.g. immunogenic fragment thereof) relating to a pathogen or toxin of interest which is intended to be protected against. The plasmid can be grown in bacteria, such as for example E. coli and then isolated and prepared in an appropriate medium, depending upon the intended route of administration, before being administered to the host. Following administration, the plasmid is taken up by cells of the host wherein the encoded peptide is produced. The plasmid vector will preferably be made without an origin of replication which is functional in eukaryotic cells, in order to prevent plasmid replication in the mammalian host and integration within chromosomal DNA of the animal concerned.
DNA vaccination can be advantageous over traditional forms of vaccination in several respects. Firstly, it is predicted that because the proteins which are encoded by the DNA sequence are synthesized in the host, the structure or conformation of the protein will be similar to the native protein associated with the disease state. It is also likely that DNA vaccination can offer protection against different strains of a virus, by generating cytotoxic T lymphocyte responses that recognize epitopes from conserved proteins. Furthermore, because the plasmids are taken up by the host cells where antigenic protein can be produced, a long-lasting immune response can be elicited. The technology also offers the possibility of combining diverse immunogens into a single preparation to facilitate simultaneous immunization in relation to a number of disease states.
Further background on DNA vaccination can be found in Donnelly J. et al, “DNA Vaccines” Annu. Rev. Immunol. 1997, 15: 617 48, the disclosure of which is included herein in its entirety by way of reference.
Accordingly, in one embodiment, the invention provides a DNA immunogenic composition, i.e. a DNA vaccine composition, comprising at least one expression vector, which may be expressed by the cellular machinery of the subject to be vaccinated or inoculated, and, optionally, a pharmaceutically acceptable excipient. The nucleotide sequence of this plasmid can encode, inter alia, one or more anthrax spore-associated immunogens (proteins) capable of inducing, in the subject to be vaccinated or inoculated, a cellular immune response (mobilization of the T lymphocytes) and/or a humoral immune response (stimulation of the production of antibodies specifically directed against the immunogen). The encoded immunogens can also be immunogenic fragments or functional variants of the anthrax spore-associated proteins as described herein. Nucleic acid-based immunogenic compositions, i.e. DNA vaccines, are described for example, in U.S. Pat. Nos. 5,589,466 and 7,074,770, the disclosures of which are hereby incorporated by reference in their entireties.
In another embodiment, the present invention provides a pharmaceutical and/or immunogenic polypeptide to the interior of a cell of a vertebrate in vivo, and a method for delivering the pharmaceutical and/or immunogenic polypeptide comprising the step of introducing a preparation comprising a pharmaceutically acceptable injectable carrier and a naked polynucleotide operatively coding for the polypeptide (e.g. anthrax spore-associated protein or immunogenic or functional variant thereof) into the interstitial space of a tissue comprising the cell, whereby the naked polynucleotide is taken up into the interior of the cell and has an immunogenic effect on the vertebrate, thereby immunizing the vertebrate against infection by B. anthracis or a toxin thereof.
The anthrax spore-associated protein polynucleotides of the various embodiments of the invention can comprise a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, and fragments thereof, shown herein.
In another aspect, the present invention is directed to immunogenic compositions comprising an anthrax spore-associated protein polynucleotide and a polynucleotide which encodes the B. anthracis PA (protective antigen) protein, referred to hereinafter as the “PA polynucleotide”, or an immunogenic fragment or functional variant thereof, referred to hereinafter as the “PA fragment polynucleotide”. The PA polynucleotide may encode a full-length mature PA protein or, alternatively, a full-length, immature PA protein which comprises a nucleotide sequence encoding a signal sequence. In one embodiment, the PA polynucleotide comprises the nucleotide sequence, SEQ ID NO:159, shown herein. The anthrax spore-associated protein and B. anthracis PA protein may, in another aspect, both be encoded by one nucleic acid sequence.
The polynucleotide may be either a DNA or RNA sequence. All forms of DNA, whether replicating or non-replicating, which do not become integrated into the genome, and which are expressible, are within the methods contemplated by the invention. When the polynucleotide is DNA, it can also be a DNA sequence which is itself non-replicating, but is inserted into a plasmid, and the plasmid further comprises a replicator (e.g. an origin of replication). The DNA may be a sequence engineered so as not to integrate into the host cell genome. The polynucleotide sequences may code for a polypeptide which is either contained within the cells or secreted therefrom, or may comprise a sequence which directs the secretion of the peptide. With the availability of automated nucleic acid synthesis equipment, both DNA and RNA can be synthesized directly when the nucleotide sequence is known or by methods which employ PCR cloning.
The anthrax spore-associated protein polynucleotide, anthrax spore-associated protein fragment polynucleotide PA polynucleotide, and PA fragment polynucleotides can be incorporated into the immunogenic compositions in one of several forms, including a linear molecule, a plasmid, a viral construct, or a bacterial construct, such as, for example, a Salmonella construct to provide a vaccine. In those cases where the immune response is elicited by administration of both the anthrax spore-associated protein polynucleotide or anthrax spore-associated protein fragment polynucleotide and the PA polynucleotide or PA fragment polynucleotide, the polynucleotides may be incorporated into separate nucleic acid molecules which are co-administered to the subject. Alternatively, the anthrax spore-associated protein polynucleotide (or anthrax spore-associated protein fragment polynucleotide) and PA polynucleotide (or PA fragment polynucleotide) may be incorporated into the same nucleic acid. In such case, the anthrax spore-associated protein polynucleotide and PA polynucleotide may be operably linked to separate promoters or to the same promoter.
In addition, the present invention contemplates pharmaceutical compositions that comprise a combination of polypeptides and polynucleotides wherein the polynucleotides can encode the polypeptides of the invention. For example, one pharmaceutical composition of the invention can comprise both a B. anthracis spore-associated polypeptide (or immunogenic or functional variant thereof) and a polynucleotide encoding B. anthracis PA (or immunogenic or functional variant thereof). Alternatively, the pharmaceutical composition can comprise at least one B. anthracis spore-associated polypeptide (or immunogenic or functional variant thereof) and a polynucleotide encoding at least one B. anthracis spore-associated protein (or immunogenic or functional variant thereof). In such pharmaceutical compositions, the polypeptide component and polypeptide component can be contained together in the same composition or each can be separately contained and provided as separate components which can be co-administered. For the purposes of this invention, “co-administering” is administration of two or more medicaments or pharmaceutical compositions (e.g. a polypeptide component and a polynucleotide component) at the same time or at about the same time, e.g. sequential administration. Sequential administration also encompasses an administration regimen occurring in some pattern over the course of days, weeks, or months, such as, for example, administering on a first day a polypeptide component followed by on a second day a polynucleotide component. There is no intended limitation on the manner in which co-administration may occur and the skilled artisan will be able to competently design a suitable co-administration regimen.
In an additional embodiment of the invention, certain modifications in the anthrax spore-associated antigens (proteins) exist, due to, for example, deletions of part of the nucleotide sequence encoding the antigen, insertions of a DNA fragment into the nucleotide sequence encoding the antigen, or into non-translated regions upstream or downstream. Such modifications may enhance the efficacy of the DNA immunogenic compositions, for example, by enhancing the level of expression of the antigen or its presentation. However, care must be taken that manipulations of the nucleotide sequence encoding the antigen do not bring about a reduction or loss of the initial immunological activity. Furthermore, the modifications carried out on the nucleotide sequence of one antigen cannot necessarily be directly transposed to another antigen, because antigens do not always have the same structural arrangements.
In one embodiment of the DNA immunogenic compositions of the invention, the expression vector can be a plasmid. The term “plasmid” covers a DNA transcription unit comprising a polynucleotide sequence comprising the sequence of the gene to be expressed and the elements necessary for its expression in vivo. In additional embodiments, the circular plasmid form, supercoiled or otherwise, or the linear form may be employed. When several genes are present in the same plasmid (e.g. the combination of a nucleotide encoding a B. anthracis spore-associated protein and a nucleotide encoding B. anthracis PA), they may be provided in the same transcription unit or in two transcription units or in several different or more transcription units. In another embodiment of the DNA immunogenic composition of the invention, the expression vector is a virus. Viral vectors appropriate for delivery of a polynucleotide sequence are known in the art.
The anthrax spore-associated protein polynucleotide or anthrax spore-associated protein fragment polynucleotide may be operably linked to a promoter which drives expression of the protein or fragment. Such promoter may be a constitutive promoter, such as, for example, the viral promoter derived from cytomegalovirus (CMV). Other viral promoters include, without limitation, CMV-IE, SV40 virus early or late promoter, and the Rous Sarcoma virus LTR promoter. Employable cellular promoters include, without limitation, that of a cytoskeleton gene, such as the desmin promoter, or, alternatively, the actin promoter. Inducible promoters are likewise contemplated, such as, for example, the lac promoter or a tissue specific promoter, such as the whey acidic protein promoter.
In one embodiment of the DNA immunogenic composition of the invention, the nucleotide sequence encoding the immunogen is in an optimized form. Optimization is understood to mean any modification of the nucleotide sequence, in particular which manifests itself at least by a higher level of expression of this nucleotide sequence, and/or by an increase in the stability of the messenger RNA encoding this antigen, and/or by the triggered secretion of this antigen into the extracellular medium, and having as direct or indirect consequence an increase in the immune response induced. Such optimization of the antigen of interest may, for example, consist in the insertion of a stabilizing intron into the gene encoding the antigen of interest to avoid the aberrant splicings of its messenger RNA and maintain the physical integrity of the latter.
In additional embodiments of the DNA immunogenic compositions of the invention, the expression vector also contains a ribosome binding site, including an internal ribosome site, for translation initiation and a transcription terminator. The vector may further include appropriate sequences for amplifying expression. In addition, expression vectors may contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance for bacterial cell cultures such as E. coli. One of ordinary skill in the art will appreciate that the particular selectable marker chosen will, like the expression vector itself, depend on the properties of the host organism.
The expression vector containing the appropriate DNA sequence(s) as hereinabove described, as well as an appropriate promoter or expression control sequence, may be employed to transform an appropriate host to permit the host to express the protein. As representative examples of appropriate host cells, there may be mentioned bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila and Sf9; animal cells such as CHO, COS or Bowes melanoma; plant cells, etc. The selection of an appropriate host for this type of recombinant polypeptide production is also within the capability of those skilled in the art from the teachings herein. Suitable expression vectors and promoters are replicable and viable in the selected host cell. The quantity of DNA used in the vaccines according to the present invention can be between about 10 micrograms and about 2000 micrograms and preferably between about 50 micrograms and about 1000 micrograms. Persons skilled in the art will have the competence necessary to precisely define the effective dose of DNA to be used for each immunization or vaccination protocol.
Introduction of the DNA vaccine vectors of the present invention into the host cell can be effected by any known method, including calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (see Davis et al., Basic Methods in Molecular Biology, (1986)). It is possible for the vectors of the present invention to be administered in a naked form (that is as naked DNA not in association with liposomal formulations, with viral vectors or transfection facilitating proteins) suspended in an appropriate medium, for example a buffered saline solution such as PBS and then injected intramuscularly, subcutaneously, intraperitonally or intravenously, although some earlier data suggests that intramuscular or subcutaneous injection is preferable (Brohm W et al, “Routes of Plasmid DNA Vaccination that Prime Murine Humoral and Cellular Immune Reponses,” Vaccine, Vol 16, No. 9/10, pp 949 954, 1998), (the disclosure of which is incorporated herein in its entirety by way of reference). It is additionally possible for the vectors to be encapsulated by, for example, liposomes or within polylactide co-glycolide (PLG) particles (Vordermeier, H. M., Coombs, A. G. A., Jenkins, P. McGee, J. P., O'Haga, D. T. Davis, S. S, and Singh, M. Synthetic delivery systems for tuberculosis vaccines: immunological evaluation of the M. tuberculosis 38 kDa protein entrapped in biodegradable PLG microparticles. Vaccine 13: 1576 1582 1995) for administration via the oral, nasal or pulmonary routes. It is also possible, according to a preferred embodiment of the invention, for intradermal administration of the vector, preferably via use of gene-gun (particularly particle bombardment) administration techniques. Such techniques may involve lyophilization of a suspension comprising the vector and subsequent coating of the vector on to gold beads which are then administered under high pressure into the epidermis, such as, for example, as described in Haynes J R. McCabe D E. Swain W F. Wedera G. Fuller J T. Particle-mediated nucleic acid immunization. Journal of Biotechnology. 44: 37 42, 1996. The amount of DNA delivered can vary significantly, depending upon the species and weight of mammal being immunized, the nature of the disease state being treated/protected against, the vaccination protocol adopted (i.e. single administration versus repeated doses), the route of administration and the potency and dose of the adjuvant compound chosen. Based upon these variables, a medical or veterinary practitioner will readily be able to determine the appropriate dosage level.
It is possible for the DNA vector, including the DNA sequence encoding the antigenic peptide, to be administered on a once off basis or to be administered repeatedly, for example, between 1 and 7 times, preferably between 1 and 4 times, at intervals between about 1 day and about 18 months. Once again, however, this treatment regime will be significantly varied depending upon the size and species of animal concerned, the disease which is being treated/protected against, the amount of DNA administered, the route of administration, the potency and dose of adjuvant compound selected and other factors which would be apparent to a skilled veterinary or medical practitioner. To enhance the immune response, the DNA vaccine compositions of the present invention can be administered with at least one adjuvant, such as those described in U.S. Pat. No. 7,074,770 which is incorporated by reference herein in its entirety. Any adjuvant compound that serves to increase the immune response induced by the antigen (either directly administered or expressed in a DNA vaccine) is contemplated by the present invention.
Formulations for injection of the DNA vaccines of the invention via, for example, the intramuscular, intraperitonile, or subcutaneous administration routes include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. Formulations suitable for pulmonary administration via the buccal or nasal cavity are presented such that particles containing the active ingredient, desirably having a diameter in the range of 0.5 to 7 microns, are delivered into the bronchial tree of the recipient. Possibilities for such formulations are that they are in the form of finely comminuted powders which may conveniently be presented either in a pierceable capsule, suitably of, for example, gelatin, for use in an inhalation device, or alternatively, as a self-propelling formulation comprising active ingredient, a suitable liquid propellant and optionally, other ingredients such as surfactant and/or a solid diluent. Self-propelling formulations may also be employed wherein the active ingredient is dispensed in the form of droplets of a solution or suspension. Such self-propelling formulations are analogous to those known in the art and may be prepared by established procedures. They are suitably provided with either a manually-operable or automatically functioning valve having the desired spray characteristics; advantageously the valve is of a metered type delivering a fixed volume, for example, 50 to 100 microliters upon each operation thereof.
i) Preparing the Anthrax Spore-Associated Protein, the PA Protein, and Fragments Thereof
The anthrax spore-associated proteins (or immunogenic and functional variants thereof) and PA proteins (or immunogenic and functional variants thereof) can be obtained by any suitable means, including, for example, purification from B. anthracis cultures or prepared as recombinant proteins from cultures of recombinant organisms. Within the context of this application, “purified” anthrax spore-associated proteins and PA proteins (and any immunogenic or function variant thereof) refers to preparations that are comprised of at least 90% anthrax spore-associated protein or PA protein, and no more than 10% of the other proteins found in the cell-free extracts or extracellular medium from which these proteins are isolated. Such preparations are said to be at least 90% pure. The PA protein may be isolated and purified from the supernatant of B. anthracis cultures using techniques known in the art, for example, as described in Methods Enzymol. 165: 103-116, 1988, which is specifically incorporated herein by reference.
In one embodiment, the anthrax spore-associated protein, PA protein, and any immunogenic fragments or functional variants thereof are prepared using recombinant techniques. Such techniques employ nucleic acid molecules which encode the anthrax spore-associated protein, the PA protein, or immunogenic fragments and functional variants thereof. For example, the proteins or fragments thereof may be produced using cell-free translation systems and RNA molecules derived from DNA constructs that encode the such proteins or fragments. Alternatively, the proteins or fragments may be made by transfecting host cells with expression vectors that comprise a DNA sequence that encodes one of the proteins or fragments and then inducing expression of the protein or fragment thereof in the host cells. For recombinant production, recombinant constructs comprising one or more of the sequences which encode the desired protein or fragment are introduced into host cells by conventional methods such as calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape lading, ballistic introduction or infection.
The desired protein or fragment is then expressed in suitable host cells, such as for example, mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters using conventional techniques, as mentioned in the preceding sections. Following transformation of the suitable host strain and growth of the host strain to an appropriate cell density, the cells can be harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification of the desired protein or fragment. In an alternative embodiment, the desired proteins or fragments thereof can be engineered with a secretory pathway signal such that the protein or desired fragments are secreted into the culture medium and obtained directly therefrom. Such secretion systems will be known in the art and will depend on the host cell in which the expression vector is being propagated in.
Conventional procedures for isolating recombinant proteins from transformed host cells are contemplated by the present invention. Such methods include, for example, isolation of the protein or fragments of interest by initial extraction from cell pellets or from cell culture medium, followed by salting-out, and one or more chromatography steps, including aqueous ion exchange chromatography, size exclusion chromatography steps, high performance liquid chromatography (HPLC), and affinity chromatography may be used to isolate the recombinant protein or fragment. Guidance in the procedures for protein purification can be found in the technical literature, including, for example, Methods in Enzymology, Volume 182, Guide to Protein Purification, Eds. J. Abelson, M. Simon, Academic Press, 1st Edition, 1990, which is already incorporated by reference.
ii) Preparing the Immunogenic Compositions
To prepare the immunogenic compositions in accordance with one of the embodiments of the invention, it is possible to use known methods of purification, synthesis, or genetic engineering. Protein fragments, naked DNA/RNA, recombinant DNA/RNA, or messenger RNA may be incorporated into pharmaceutical compositions appropriate for the anticipated method of administration, such as excipients.
Various genetically engineered virus hosts, i.e. recombinant viruses, can be used to prepare anthrax spore-associated protein (and, optionally, PA) immunogenic compositions. Examples of recombinant virus hosts include, without limitation, vaccinia virus, recombinant canarypox, and defective adenovirus. Other suitable viral vectors include retroviruses that are packaged in cells with amphotropic host range and attenuated or defective DNA virus, such as herpes simplex virus, papillomavirus, Epstein Barr virus, and adeno-associated virus.
In one embodiment, adjuvants may be used to enhance the effectiveness of the immunogenic compositions of the invention. The term “adjuvant” as used herein refers to a compound or mixture which enhances the immune response to an antigen. Desirable characteristics of ideal adjuvants include, without limitation, lack of toxicity, ability to stimulate a long-lasting immune response, simplicity of manufacture and stability in long-term storage, synergy with other adjuvants, capability of selectively interacting with populations of antigen presenting cells (APC), ability to specifically elicit appropriate THH1 or TH2 cell-specific immune responses, and ability to selectively increase appropriate antibody isotype levels (for example IgA) against antigens.
Exemplary adjuvants include, without limitation: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations (WO 90/14837; WO 99/30739); (3) saponin adjuvants, such as Stimulon™ (Cambridge Bioscience, Worcester, Mass.) or particles generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA); (5) cytokines, such as interleukins (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-16, IL-17, IL-19, IL-20, and the like), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), VEGF, CD27, CD30, CD40, Fas Ligand, Placenta Growth Factor, etc.; (6) detoxified mutants of a bacterial ADP-ribosylating toxin such as a cholera toxin (CT), a pertussis toxin (PT), or an E. coli heat-labile toxin (LT), adjuvants derived from the CpG family of molecules; (7) R-848 (U.S. Pat. No. 5,352,784; WO99/29693); and (8) other substances that act as immunostimulating agents to enhance the effectiveness of the composition.
The determination of the amount of the respective components included in certain embodiments of the immunogenic compositions of the invention, such as antigen, lipoprotein, and adjuvant, as well as the preparation of those compositions, can be in accordance with standard techniques well known to those skilled in the pharmaceutical or veterinary arts. In particular, the afore-mentioned amounts and the dosages administered are determined taking into consideration such factors as the particular antigen, the lipoprotein, the adjuvant, the age, sex, weight, species and condition of the particular patient, and the route of administration.
The immunogenic compositions of the invention may be formulated by dispersing anthrax spore-associated protein (and any immunogenic fragments thereof or functional variants thereof) and, optionally, rPA or PA in the desired amount in any pharmaceutical carrier suitable for use in vaccines. Typical doses of anthrax vaccine are 0.5 mL in volume, but any volume suitable to deliver the desired amount of anthrax spore-associated protein (or any immunogenic fragments or functional variants thereof) and PA, if applicable, can be used. Any pharmaceutical excipient suitable for administration to mammals which does not interfere with the immunogenicity of the anthrax spore-associated protein (and PA, if applicable) may be employed. Example excipients include, without limitation, sterile water, physiological saline, glucose or the like.
The immunogenic compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “REMINGTON'S PHARMACEUTICAL SCIENCE”, 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.
Compositions of the invention may be provided as liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions or viscous compositions, which may be buffered to a selected pH. If digestive tract absorption is preferred, compositions of the invention can be in the “solid” form of pills, tablets, capsules, caplets and the like, including “solid” preparations which are time-released or which have a liquid filling, e.g., gelatin covered liquid, whereby the gelatin is dissolved in the stomach for delivery to the gut.
If nasal or respiratory (mucosal) administration is desired, compositions may be prepared as inhalables, sprays, and the like and dispensed by a squeeze spray dispenser, pump dispenser, or aerosol dispenser. Aerosols are usually under pressure by means of a hydrocarbon. Pump dispensers can preferably dispense a metered dose or, a dose having a particular particle size.
Compositions within the scope of this invention can contain a humectant to inhibit drying of the mucous membrane and to prevent irritation. Any of a variety of pharmaceutically acceptable humectants can be employed including, for example sorbitol, propylene glycol or glycerol. As with the thickeners, the concentration will vary with the selected agent, although the presence or absence of these agents, or their concentration, is not an essential feature of this invention.
Enhanced absorption across the mucosal and especially nasal membrane can be accomplished employing a pharmaceutically acceptable surfactant. Typically useful surfactants for compositions include polyoxyethylene derivatives of fatty acid partial esters of sorbitol anhydrides such as Tween 80, Polyoxynol 40 Stearate, Polyoxyethylene 50 Stearate and Octoxynol.
A pharmaceutically acceptable preservative can be employed to increase the shelf-life of the compositions. Benzyl alcohol may be suitable, although a variety of preservatives including, for example, Parabens, thimerosal, chlorobutanol, or benzalkonium chloride may also be employed.
Compositions of the invention can contain pharmaceutically acceptable flavors and/or colors for rendering them more appealing, especially if they are administered orally. The viscous compositions may be in the form of gels, lotions, ointments, creams and the like and will typically contain a sufficient amount of a thickening agent so that the viscosity is from about 2500 to 6500 cps, although more viscous compositions, even up to 10,000 cps may be employed. Viscous compositions can be formulated within the appropriate viscosity range to provide longer contact periods with mucosa, such as the lining of the stomach or nasal mucosa.
The choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form [e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form, or solid dosage form [e.g., whether the composition is to be formulated into a pill, tablet, capsule, caplet, time release form or liquid-filled form].
Solutions, suspensions and gels, normally contain a major amount of water (preferably purified water) in addition to the antigen and other optional components. Minor amounts of other ingredients such as pH adjusters (e.g., a base such as NaOH), emulsifiers or dispersing agents, buffering agents, preservatives, wetting agents, jelling agents, (e.g., methylcellulose), colors and/or flavors may also be present. The compositions can be isotonic, i.e., it can have the same osmotic pressure as blood and lacrimal fluid.
The desired isotonicity of the compositions of this invention may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is preferred particularly for buffers containing sodium ions.
Viscosity of the compositions may be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose is preferred because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The preferred concentration of the thickener will depend upon the agent selected. The important point is to use an amount which will achieve the selected viscosity. Viscous compositions are normally prepared from solutions by the addition of such thickening agents.
A pharmaceutically acceptable preservative can be employed to increase the shelf-life of the compositions. Benzyl alcohol may be suitable, although a variety of preservatives including, for example, parabens, thimerosal, chlorobutanol, or benzalkonium chloride may also be employed. A suitable concentration of the preservative will be from 0.02% to 2% based on the total weight although there may be appreciable variation depending upon the agent selected.
Those skilled in the art will recognize that the components of the compositions can be selected to be chemically inert with respect to the antigen and other optional components. This will present no problem to those skilled in chemical and pharmaceutical principles. The skilled person in view of problems encountered in the formulation of the medicaments of the invention can readily reference standard technical texts or carry out experimentation which is not undue to determine the best and most appropriate manner to formulate the medicaments of the invention.
The immunologically effective compositions of this invention are prepared by mixing the ingredients following generally accepted procedures. For example the selected components may be simply mixed in a blender, or other standard device to produce a concentrated mixture which may then be adjusted to the final concentration and viscosity by the addition of water or thickening agent and possibly a buffer to control pH or an additional solute to control tonicity as in manners exemplified but not limited to the above description.
In additional embodiments, the invention is directed to methods of using the nucleic acid-based or protein-based immunogenic compositions described above to elicit a protective immune response against lethal infection with B. anthracis or its toxins in an animal subject. The method comprises administering one of the above-described immunogenic compositions to the subject in a therapeutically effective amount. As used herein, the term “therapeutically effective amount” can mean that the amount administered can have a protective effect against pathologic consequences of infection, i.e. a therapeutic benefit. The compositions can be administered at a dosage sufficient to elicit, prime, or boost an immune response which prophylactically protects against a lethal B. anthracis infection in the animal. The animal subject may be any mammal, including a human subject. The immune response prophylactically prevents a lethal B. anthracis infection in the animal. The active immunity elicited by immunization with the above-described immunogenic compositions can prime or boost a cellular or humoral immune response.
Immunogenic compositions according to the invention may be administered to a subject in which it is desired to elicit an immune response against B. anthracis. In addition to humans, the compositions of the present invention may advantageously be administered, for example, to horses, cattle, oxen, goats, sheep, dogs, cats, antelope, buffalo, rabbits, pigs, and the like.
In one embodiment, the method of the invention comprises directly administering a nucleic acid, particularly a DNA, which encodes at least one anthrax spore-associated protein, an immunogenic fragment thereof, a functional variant thereof and optionally, PA or immunogenic and/or functional variant fragments thereof, into the subject. In another embodiment, the protein or peptide-based immunogenic compositions of the invention are administered to the animal subject.
Administration may be made in a variety of routes including, without limitation, orally, transbucally, transmucosally, sublingually, nasally, rectally, vaginally, intranasally, intraocularly, intramuscularly, intralymphatically, intravenously, subcutaneously, transdermally, intradermally, intra tumor, topically, transpulmonarily, by inhalation, by injection, or by implantation, etc. In one embodiment, the nucleic acid-based composition of the invention is introduced into muscle tissue; in other embodiments, the nucleic acid-based composition is incorporated into tissues of skin, brain, lung, liver, spleen or blood. The preparation may be placed within cavities of the body. In still other embodiments, the nucleic acid based-composition is impressed into the skin or administered by inhalation.
Means of administration further include, without limitation, gold particles coated with DNA and projected so as to penetrate into the cells of the skin of the subject to be vaccinated (Tang et al. Nature 1992.356.152-154) and the liquid jet injectors which make it possible to transfect both skin cells and cells of the underlying tissues (Furth et al. Analytical Bioch. 1992. 205.365-368).
Those skilled in the art will recognize that for injection, formulation in aqueous solutions, such as Ringer's solution or a saline buffer may be appropriate. Liposomes, emulsions, and solvents are other examples of delivery vehicles. Oral administration would require carriers suitable for capsules, tablets, liquids, pills, etc, such as sucrose, cellulose, etc.
Dosage treatment may be a single dose schedule or a multiple dose schedule. A multiple dose schedule can be one in which a primary course of vaccination may be with 1 dose, followed by another dose given at a subsequent time interval, chosen to maintain and/or reinforce the immune response. The 1 or 2 injections may be carried out over an extended period of time. Thus, in one embodiment of the immunogenic compositions of the invention, a desired anti-anthrax spore-associated protein (and, optionally, anti-PA) antibody titer is obtained in a subject with fewer doses of the immunogenic composition than the regimen employed with AVA: six doses administered over 18 months. In another embodiment, the method of the invention involves administration of 1 or 2 doses to obtain a desired anti-anthrax spore-associated protein (and, optionally, anti-PA) antibody titer in an immunized mammalian subject such as a human. In yet another embodiment, protective immunity to B. anthracis is imparted to the immunized subject.
Anti-anthrax spore-associated protein or anti-PA titer, measured as the reciprocal of the dilution of serum at which no anthrax spore-associated protein-reactive or PA-reactive antibody, respectively, is detected, is a common measure of the effectiveness of anthrax vaccines. (Pittman et al., Vaccine, 19:213-216 (2000)). The interval between repeated administrations of the immunogenic composition may vary, and judicious spacing of the doses can increase the immune response, as measured by anti-anthrax spore-associated protein or anti-PA titer. Any spacing of doses may be employed that achieves the desired immune response.
The immunogenic compositions of the invention may be administered in a dosage sufficient to prevent a lethal B. anthracis infection in a subject through a series of immunization challenge studies using a suitable animal host system, e.g. rhesus macaques, which are thought to be an acceptable standard for human use considerations. The dosage regimen will also, at least in part, be determined by the need of the subject and be dependent on the judgment of the clinician. The dosage to be administered depends on the size of the subject being treated as well as the frequency of administration and route of administration. Ultimately, the dosage will be determined using clinical trials. Initially, the clinician will administer doses that have been derived from animal studies. If prevention of disease is desired, the vaccines can generally be administered prior to primary infection with the pathogen of interest. If prevention of disease post-infection (but before germination of spores) or prevention of progression of disease, e.g., the reduction of symptoms or recurrences after infection and germination of spores, is desired, the vaccines can generally be administered within about one to about sixty days after primary infection, or after primary infection in concert with other anti-anthrax treatment, respectively.
For any composition to be administered to an animal or human, including the components thereof, and for any particular method of administration, it is preferred to determine therefor: toxicity, such as by determining the lethal dose (LD) and LD50 in a suitable animal model e.g., rodent such as mouse; and, the dosage of the composition(s), concentration of components therein and timing of administering the composition(s), which elicit a suitable immunological response, such as by titrations of sera and analysis thereof for antibodies or antigens, e.g., by ELISA. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure and the documents cited herein. As discussed above, the time frame for sequential administrations can be ascertained without undue experimentation.
The present invention also contemplates antibodies against the antigens of the invention, for example, the anthrax spore-associated proteins of the invention, and any immunogenic fragments thereof or functional variants thereof, and any suitable methods for preparing the antibodies that are available to the skilled artisan. The antibodies can be used in diagnostic methods for detecting infections of B. anthracis or the presence of B. anthracis toxins and for treating infections of B. anthracis.
Antibodies that bind the anthrax spore-associated proteins, and PA, and any immunogenic and/or functional variants thereof can be prepared by a variety of methods that are known in the art and outlined in the technical literature, for example, in Current Protocols in Molecular Biology, Ausubel, F. M. et al., (eds.) Greene Publishing Associates, (1989), Chapter 2. As one example of such methods, a preparation of an anthrax spore-associated protein of the invention or immunogenic and/or functional variant thereof is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.
Monoclonal antibodies specific for the proteins of the invention, or immunogenic and/or functional variants thereof can be prepared using hybridoma technology (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)). In general, an animal (e.g. a mouse) is immunized with a protein of the invention. The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, for example, the parent myeloma cell line (SP2O), available from the ATCC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232 (1981)). The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding a the proteins of the invention, e.g. the anthrax spore-associated proteins or immunogenic and/or functional variants thereof of the invention.
Alternatively, additional antibodies capable of binding to proteins of the invention can be produced in a two-step procedure using anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and therefore, it is possible to obtain an antibody which binds to a second antibody. In accordance with this method, protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the protein of the invention-specific antibody can be blocked by the protein of the invention. Such antibodies comprise anti-idiotypic antibodies to the protein of the invention-specific antibody and are used to immunize an animal to induce formation of further protein of the invention-specific antibodies.
For in vivo use of antibodies in humans, an antibody can be “humanized”. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric and humanized antibodies are known in the art and are discussed herein. (See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., International Publication No. WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985)), each of which are incorporated by reference in their entireties.
The present invention further contemplates diagnostic methods which use the antibodies of the invention, e.g. those directed against the anthrax spore-associated proteins or immunogenic fragments and/or functional variants thereof, to diagnose an infection of B. anthracis or the presence of a B. anthracis toxin. In one aspect, the present invention contemplates an immunoassay that tests a subjects blood or tissues using the antibodies of the invention to detect or determine whether the blood or tissue comprises B. anthracis spores, whole bacteria, or toxins thereof. The antibodies can be provided in the form of a diagnostic kit, which can include other necessary or desirable components, such as sterile vessels for reacting the blood/tissue with the antibodies, antibodies, syringes or other advantageous implements or instruments, and any necessary or desirable reagents.
The instant invention further contemplates pharmaceutical compositions comprising the antibodies of the invention in a therapeutically effective dose or quantity and any desirable or advantageous excipients. Pharmaceutical compositions have been described above. The pharmaceutical compositions comprising the antibodies of the invention can be administered to a subject in need thereof, e.g. a patient or animal infected with B. anthracis, by any means known to the skilled artisan and as described herein.
In one embodiment, the invention provides kits containing the immunogenic compositions of the invention and instructions for admixture and/or administration. The kits can comprise the polypeptide-based compositions (e.g. a therapeutically effective dose of an anthrax spore-associated protein of the present invention, or an immunogenic fragment or functional variant thereof), or nucleic-acid compositions (e.g. a nucleotide vector encoding an anthrax spore-associated protein of the invention, or an immunogenic fragment or functional variant thereof), or a combination of both. In the case of the combination, the kit can comprise separate vessels of the polypeptide-based compositions and the nucleic-acid based compositions or alternatively, such compositions can be combined together in a suitable admixture. In one embodiment, the invention provides a kit comprising an immunogenic composition comprising at least one anthrax spore-associated protein or an immunogenic composition comprising at least one expression vector, wherein the expression vector contains a nucleic acid molecule encoding an anthrax spore-associated protein or fragment thereof and instructions for administering the immunogenic composition to induce an immunological response in a subject.
The kits contemplated by the invention can also contain any implement for the successful and complete delivery of the compositions of the invention, such as, but not limited to, a syringe, sterile mixing vessel, measuring device, and instructions, etc. The kits of the invention are also not limited to the provision of a single dose or delivery of the compositions of the present invention, but can contain any suitable quantity of doses, such as, a suitable quantity of compositions to last 1 week, 1 month, or 1 year or more.
Any of the compositions of the kits of the invention can also include other suitable polypeptides or polypeptide-encoding nucleotide vectors of the invention, such as B. anthracis PA or an immunogenic fragment or functional variant thereof.
While the description above refers to particular embodiments of the present invention, it will be understood that many modifications may be made without departing from the spirit thereof. The accompanying claims are intended to cover such modifications as would fall within the true scope and spirit of the present invention.
The present invention is additionally described by way of the following illustrative, non-limiting Examples that provide a better understanding of the present invention and of its many advantages.
An inducible, B. anthracis genomic DNA expression library was first constructed using genomic DNA isolated from the non-pathogenic B. anthracis Sterne strain in the pET30 (abc) series of expression vectors (which permit cloning of inserts in each of three reading frames under the control of the T7 phage promoter), and the expression host E. coli BL21 (DE3) (Novagen, Madison, Wis.). A limited expression library of putative anthrax spore-surface (spore-associated) proteins was then generated by screening the above genomic expression library with affinity-purified, polyclonal antibodies generated against a mixture of gamma-irradiated, purified, intact spores produced by B. anthracis Vollum, Ames and Sterne strains, in goats (Chemicon, Temecula, Calif.). A total of 292 reactive clones were identified (unpublished data), and comprised the limited, expression library of anthrax spore-associated proteins that was probed with sera from AVA-vaccinated humans (see below) in this study.
Pre-immune and immune serum samples were collected from two human adult volunteers immunized with AVA at the Division of Infectious Diseases, Massachusetts General Hospital, Boston, Mass. The institutional review board (IRB) of the Massachusetts General Hospital approved the collection and use of these serum samples. Specifically, serum samples (10 ml) were collected prior to the first administration (pre-immune) and two weeks following the fourth administration (dose administered at six months) of AVA (immune sera). Sera from this time point were utilized as a probe for the screen, since results of experiments in non-human primates indicate that protective immunity against inhalational anthrax is engendered following two administrations of AVA (Friedlander, A. M., et al., 1999. JAMA 282:2104-2106). Serum samples were dispensed in small volumes and stored at −70° C. until used.
Prior to use as probes, sera were pooled to compensate for variations in immune responses of individuals and to identify a wider array of reactive spore-associated proteins, and were used either directly (crude sera) or following affinity purification (affinity-purified sera). Sera were affinity-purified using magnetic beads linked to either Protein A or Protein G (Dynabeads Protein A or Dynabeads Protein G, respectively), as per the instructions of the manufacturer (Dynal Biotech, Lake Success, N.Y.), with modifications. Protein A reportedly binds all human immunoglobulin (Ig) isotypes and IgG subclasses except IgG3, whereas Protein G binds all IgG subclasses but not other Ig isotypes (Ed Harlow and David Lane. 1988. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Initially, pooled sera were affinity-purified using both Dynabeads Protein A and Dynabeads Protein G; however, pilot colony immunoblotting experiments revealed that the pooled sera affinity purified using Dynabeads Protein A consistently yielded better results (data not shown), and this affinity-purified sera was therefore used as a probe in subsequent colony immunoblotting experiments.
For capture of antibodies by Dynabeads Protein A, 10 μl of pooled pre-immune or immune sera was added to 100 μl of beads, prepared as instructed by the manufacturer, and incubated at room temperature with slow tilt rotation for 30 min. The beads were then pulled down using a magnet, the supernatant decanted, and beads washed as instructed by the manufacturer to remove loosely bound components. Specifically bound Igs were eluted with 0.1 M citrate (pH 3.0) directly into 1 M Tris (pH 9.0). Crude and affinity-purified sera were stored at 4° C. following the addition of 0.02% sodium azide until further use. Long-term storage was in 50% glycerol at −70° C.
Because human-use acellular PA-based vaccines were found to induce weak and inconsistent immune responses (Hambleton, P., et al., 1984. Vaccine 2:125-13229; Lincoln, R. and D C Fish. 1970. Anthrax toxin, p. 361-414. In T. C. Monte, et al., Academic Press, Inc., New York), and due to the lack of standardization of the vaccine manufacturing process (Leppla, S. et al., 2002. J. Clin. Invest. 110:141-144), the quality of pooled, immune sera was examined prior to use as a probe for screening the previously generated limited expression library of anthrax spore-associated proteins. The quality of crude and affinity-purified sera was assessed by reacting pooled, pre-immune and immune sera with a recombinant (test) clone, E. coli BL21 (DE3)(pSMR-PA), expressing full-length PA utilizing a colony immunoblot assay. Reactivity against this particular protein was examined, since PA reportedly is the principal immunogen and a major component of AVA (Leppla, S. H., et al, J. Clin. Invest. 110:141-144), and anti-PA antibodies are a gauge of the host response to immunization (Joellenbeck, L. M., et al., 2002. National Academy Press, Washington, D.C.).
For immunoscreening, the test clone and E. coli BL21 (DE3) (pET30a) (negative control) were tooth-picked on duplicate Luria-Bertani (LB) plates supplemented with 50 μg/ml of kanamycin (LB-Kan) and incubated overnight at room temperature. Colonies were lifted from one of the plates (the other plate constituted the “Master” plate) using a nitrocellulose filter and placed colony side up on a fresh LB-Kan plate containing 1 mM isopropyl-β-D-thiogalactoside (IPTG). Following an overnight incubation at 30° C. to induce expression of genes contained within cloned inserts, colonies on plates were partially lysed by exposing them to chloroform vapors for 15 min in a candle jar. The filters were then removed from the plates, air dried, and blocked using 5% non-fat milk in phosphate buffered saline (pH 7.4) (PBS) for 1 h at room temperature. After rinsing with PBS containing 0.05% Tween 20 (PBS-T), filters were probed with a 1:5,000 dilution of either pooled, crude pre-immune or immune sera, or with a 1:500 dilution of either pooled, affinity-purified pre-immune or immune sera. Following an overnight incubation at 4° C. on a rocking platform, filters were washed 3× with PBS-T, and incubated with a 1:20,000 dilution of peroxidase-labeled goat IgG raised against the human gamma globulin fraction (ICN/Cappel, Aurora, Ohio). Filters were developed using an ECL chemiluminescence kit (Amersham Biosciences), and positive clones were identified by their positions on the “Master” plate.
As shown in
It was previously reported that unidentified antigens might significantly contribute to the protective immunity of PA-based vaccines (Brossier, F., et al., 2002. Infect. Immun. 70:661-664; Cohen, S. I., et al., 2000. Infect. Immun. 68:4549-4558; Little, S. F. and G. B. Knudson. 1986. Infect. Immun. 52:509-512; Pezard, C., et al., 1995. Infect. Immun. 63:1369-1372; Stepanov, A., et al., 1996. J. Biotechnol 44:155-160; Welkos, S., et al., 2001. Microbiology 147:1677-1685). It was also documented that immunization with AVA induces protective immunity against both cutaneous (Joellenbeck, L. M., et al., 2002. National Academy Press, Washington, D.C.; Leppla, S. H., et al., 2002. J. Clin. Invest. 110:141-144) and inhalational anthrax (Friedlander, A. M., et al., 1999. JAMA 282:2104-2106; Joellenbeck, L. M., et al., 2002. National Academy Press, Washington, D.C.; Leppla, S. H., et al., 2002. J. Clin. Invest. 110:141-144), albeit following multiple administrations. It was, thus, investigated whether a subset of 292 library clones expressing anthrax spore-surface proteins, identified might be part of the B. anthracis immunome in patients immunized with AVA. The reactivity of recombinant clones was examined by expressing these proteins with pooled, crude and affinity-purified pre-immune and immune sera from two human adult volunteers administered four doses of AVA.
Prior to screening, each of the 292 clones expressing spore-associated proteins was tooth-picked on duplicate LB-Kan plates in a grid pattern alternating with the negative control, and incubated at 37° C. for 6 h. Colonies were lifted, and induction of gene expression from cloned inserts was performed as described above. The filters were processed as detailed earlier and probed with a 1:10,000 dilution of pooled, crude pre-immune or immune sera at 37° C. for 1 h. Filters were then washed 3× with PBS-T, and incubated with a 1:20,000 dilution of peroxidase-labeled goat IgG raised against human gamma globulin fraction (ICN/Cappel) for 1 h at 37° C. Filters were washed and developed as before, and reactive clones were identified by their positions on the “Master” plate. Positive clones were purified and reactivity confirmed via an additional round of colony immunoblotting using pooled, affinity-purified pre-immune and immune sera at a dilution of 1:500 using the same procedure described in the previous section for screening the test clone and the negative control. This immunological screen resulted in the identification of 69 expression library clones expressing proteins that were targets of AVA-induced immunity.
Identification of Anthrax Spore-Associated Proteins Reactive with Immune Sera.
To identify proteins expressed from each clone, lysates of each positive clone were prepared as described earlier (Kudva, I. T., et al., 2002. J. Bacteriol. 184:1873-18791) and used as a template in PCR. Amplification reactions were performed using vector-specific primers obtained from the DNA Synthesis Core Facility, Department of Molecular Biology, Massachusetts General Hospital as described earlier (Kudva, I. T., et al., 2002. J. Bacteriol. 184:1873-1879). Amplicons were purified using the QIAQuick PCR Purification Kit (Qiagen, Valencia, Calif.) and subjected to DNA sequencing at the DNA Sequencing Core Facility, Department of Molecular Biology, Massachusetts General Hospital, using an ABI Prism DiTerminator cycle sequencing with AmpliTaq DNA polymerase FS with an ABI 377 DNA sequencer (Perkin-Elmer Applied Biosystems Division, Foster City, Calif.).
Genes on cloned inserts within reactive clones were identified via BLAST by comparing nucleotide sequences against those contained in the non-redundant database at the National Center for Biotechnology information (NCBI), and against sequences of B. anthracis Ames strain in the database at The Institute for Genomic Research (TIGR). Protein identities and functions (Table 1) were determined from the TIGR database and Swiss-Prot/trEMBL databases, or the NCBI's Conserved Domain Database (CDD; Marchler-Bauer, A. and S. H. Bryant. 2004. Nucleic Acids Res. 32:W327-W331).
B. anthracis
B. anthracis
3682
1032
3732
8122
2432
8292
3412
3872
9252
1Functional categories are based on The Institute of Genomic Research (TIGR) database grouping of proteins of the sequenced B. anthracis Ames strain.
2Two genes present on the same cloned insert.
3Encoded by a gene with no significant homology to database entries.
4Functions of identified proteins are as designated in the TIGR database or/and in Swiss-Prot.
5Putative functions of conserved hypothetical proteins determined using the Conserved Domain Database (CDD)
The anthrax spore immunome in vaccinated humans comprised of several proteins involved in protein synthesis, modification and repair (Table 1). Included within this group were clones expressing a glutamyl-tRNA synthetase (GltS), and a seryl-tRNA synthetase (SerS), both of which catalyze the attachment of specific amino acids to cognate tRNAs (tRNA aminoacylation). Of note, tRNA synthetases reportedly are present on the anthrax spore-surface (Liu, H., et al., 2004. J. Bacteriol. 186:164-178) although the precise function of such proteins in this location is unclear. Also identified was a clone expressing a polypeptide deformylase, Def-1. The deformylation it catalyzes of polypeptide chains is imperative for protein maturation, which in turn is essential for bacterial cell viability.
One protein expressed by a clone in this group was an unique RNA binding protein called SmpB, which binds with high affinity to a tmRNA molecule (functions both as a tRNA and a mRNA) encoded by ssrA (SsrA RNA) (Karzai, A. W., et al., 1999. EMBO J. 18:3793-3799) to form a complex that functions in ridding the bacterial cell of incompletely synthesized, nascent polypeptides. SmpB as a spore-associated protein may play a role in the virulence of B. anthracis. Because bacterial cells lacking tmRNA demonstrate increased sensitivity to inhibitors of protein synthesis (de la Cruz, J. and A. Vioque. 2001. RNA 7:1708-1716), SmpB may also have potential as a target for drug design. Another protein identified was the peptide chain release factor I (PrfA), a small protein that directs termination of translation in response to stop codons.
Transport and binding proteins included components of the ATP-binding cassette (ABC) superfamily, as well as members of the major facilitator superfamily (MFS). Specifically identified in this study were clones expressing components of several ABC-type transporters involved in the uptake and transport of oligopeptides. Such proteins function in Gram positive bacteria in sensing extracellular signaling molecules essential for the initiation of competence and sporulation in B. subtilis (Perego, M., et al., 1991. Mol. Microbiol. 5:173-185, Rudner D Z, et al., 1991. J. Bacteriol. 173:1388-1398), and promoting growth of Listeria monocytogenes at low temperatures and within macrophages (Borezee, E., et al., 2000. Infect. Immun. 68:7069-7077). Also identified was a clone expressing a sugar transporter (specific substrate unknown) that belonged to the MFS and another clone expressing an efflux transporter of the EamA-type, which, in E. coli, serves to regulate the level of metabolites by effluxing excess metabolites of the cysteine pathway out of the cell, which would otherwise disrupt metabolism (Franke, I., A. et al., 2003. J. Bacteriol. 185:1161-1166).
Two conserved hypothetical proteins were encoded by genes on inserts within clone #373 and clone #1077, both of which were predicted by the CDD to have S-adenosylmethioinine (SAM)-dependent methyl transferase activity. Rounding off this group was a clone expressing an integral membrane protein of the sodium:alanine symporter family (SAF). Although L-alanine is a documented spore germinant (Ireland, J. A. W. and P. C. Hanna. 2002. J. Bacteriol. 184:296-1303; Titball, R. W. and R. J. Manchee. 1987. J. Appl. Bacteriol. 62:269-273), it is currently unclear whether this symporter plays a role in spore germination following host infection.
Cell envelope proteins included orthologs of proteins implicated in the pathogenesis of other Gram positive organisms. The screen identified clones expressing proteins possessing the C-terminal LPXTG motif (SEQ ID NO: 161), a sorting signal that anchors proteins to the cell-envelope through the action of a membrane-bound cysteine protease called sortase (Lee, V. T. and O, Schneewind. 2001. Genes & Dev. 15:1725-1752). Cell-wall anchored proteins reportedly contribute to virulence of Gram positive pathogens (Xu, Y., et al., 2004. J. Biol. Chem. 279:51760-51768) and may also play a role in B. anthracis virulence. The screen identified a clone expressing a putative internalin (InlA) protein (two paralogs, namely, BA1346 and BA0552, are present in the sequenced B. anthracis Ames strain). Such spore-associated proteins may facilitate heretofore unidentified interactions between the anthrax spore and its environment, and, therefore, are likely candidates for both vaccine and drug development.
Two other clones expressing LPXTG-domain (SEQ ID NO: 161) containing proteins were also identified. The open reading frame of one of these (BAS5205/BA5604) was disrupted, but nevertheless included a collagen-binding domain. Since collagen is a primary component of the mammalian extracellular matrix, such proteins could facilitate attachment and interaction of vegetative bacilli or spores to host connective tissues. The other LPXTG-containing (SEQ ID NO: 161) protein contained a domain that is found in the vicinity of Fe3+ siderophore transporters called the “NEAT” (near transporter repeat) domain (Andrade, M. A., et al, 2002. Gen. Biol. 3:RESEARCH0047). Because of the association of NEAT domains with transporters functioning in iron acquisition and transport, a requisite for survival within the mammalian host, such proteins may play a major role in disease pathogenesis. Two clones identified expressing cell envelope proteins were an UDP-N-acetylglucosamine 1-carboxyvinyltransferase 2 (MurA2) essential for the conversion of UDP-N-acetyl glucosamine into precursors for murein for peptidoglycan cell wall biosynthesis (Bernhardt, T. G., et al., 2001. Science 292:2326-2329) and a putative glucose-1-phosphate thymidylyltransferase involved in the synthesis of deoxy-thymidine diphosphate (dTDP)-L-rhamnose, a precursor of L-rhamnose, which is a component of surface structures of both Gram positive and Gram negative bacteria such as cell wall and capsular antigens known to modulate virulence and mediate attachment to host tissues (Blankenfeldt, W. M., et al., 2000. EMBO J. 19:6652-6663). Also identified was a clone expressing a predicted membrane protein, PfoR, related to membrane components of the fructose and sucrose-specific phosphotransferase systems. BLAST analysis revealed the presence of orthologs in both B. cereus and B. thuringiensis that were annotated as possible regulatory proteins. As a spore-associated protein, PfoR may have a role in spore-germination.
The screen identified a clone expressing alanine racemase, a component of the surface of anthrax spores, as well as spores produced by other members of the B. cereus family (Steichen, C. P., et al., 2003. J. Bacteriol. 185:1903-1910). This enzyme may influence the rate of spore germination (Kanda-Nambu, K. et al., 2000. Amino Acids 18:375-387) and act in concert with other proteins to contribute to the pathogenesis of anthrax. One reactive clone expressed one of the two paralogs in the genome of the sequenced B. anthracis Ames Strain (Read, T. D., et al., 2003. Nature 423:81-86), annotated as the immune inhibitor A metalloprotease (InhA), a secreted zinc-dependent metalloprotease that is also produced by other members of the B. cereus family, and is a component of the exosporium of the B. cereus spore (Charlton, S., et al., 1999. J. Appl. Microbiol. 87:241-245). InhA in B. anthracis may function in a manner similar to that in B. thuringiensis (Dalhammar, G. and H. Steiner. 1984. Eur. J. Biochem. 139:247-252) to inactivate bactericidal host proteins during early infection and facilitate bacterial survival within the host. InhA may, in fact, be part of a suite of proteins that contribute to protective immunity against anthrax. Also included in this group were two clones expressing putative membrane proteins of unknown function, which merit further evaluation as virulence determinants in view of their surface-location.
The screen identified two clones expressing proteins involved in sporulation. Identified proteins included a Spo0B-associated GTP binding protein of the Obg family and the RNA polymerase sigma-27 factor (SigK). Also identified were reactive clones expressing proteins involved in metabolism, such as the flavoprotein subunit of the membrane bound enzyme, succinate dehydrogenase (SdhA), an enzyme of the tricarboxylic acid cycle, which during aerobic growth converts succinate to fumarate. Fumarate reductase reportedly facilitates H. pylori colonization of the murine gastric mucosa, and hence has been proposed to be both a novel drug target and a putative vaccine candidate (Ge, Z., et al., 2000. Microb. Pathog 29:279-287). Of note, the B. subtilis SdhA has also been demonstrated to function as a fumarate reductase (Schnorpfeil, M., et al., 2001. Eur. J. Biochem. 268:3069-3074).
The screen identified several clones expressing proteins involved in the metabolism of macromolecules and energy. Also identified was an enzyme involved in DNA replication, recombination and repair called endonuclease IV. As an anthrax spore-surface protein, endonuclease IV may function in concert with other proteins to facilitate spore survival within macrophages. One clone expressed a putative glutathionylspermidine (GSP) synthase, an important intermediate in the biosynthesis of the antioxidant, tryptathione. A clone expressing the acetoin utilization protein, AcuC, which facilitates the utilization of the carbon storage compound, acetoin, via an undefined mechanism, was identified. Another clone contained an insert that included three genes. The first encoded an enzyme called acyl CoA dehydrogenase (ACDH) functioning in fatty acid and phospholipid metabolism and may be an important component of the stress response functioning in conjunction with other overlapping proteins to facilitate pathogen adaptation to the in vivo environment. The second gene on the insert encoded a cytoplasmic, conserved hypothetical protein, and the third gene encoded acetyl-CoA acetyltransferase, an enzyme involved in fatty acid and phospholipid metabolism. Of note, acetyl-CoA acetyltransferase is located on the anthrax spore-surface (Liu, H., et al., 2004. J. Bacteriol. 186:164-178). Also expressed from one of the clones in this group was an enolase functioning in glycolysis/gluconeogenesis. This enzyme is a component of the anthrax spore-surface (Liu, H., et al., 2004. J. Bacteriol. 186:164-178), and was recently reported to be a component of anthrax vaccine approved for human use in the UK (Whiting, G. C., et al., 2004. Vaccine 22:4245-4251).
Several reactive clones expressing proteins involved in amino acid biosynthesis were identified. Among the proteins expressed by such clones was methylribose kinase (MtnK) (identified twice), an enzyme that is unique to microbes (and plants) and plays a central role in the salvage of methionine (Sekowska, A., et al., 2001. BMC Microbiol 1:1570, Gianotti, A. J., et al., 1990. J. Biol. Chem. 265:831-837). Anthrax spore-associated MtnK may be a suitable target for the development of vaccines, drugs, and/or spore-inactivation agents. A functional ortholog of the autoinducer synthase, LuxS, responsible for the final step of AI-2 synthesis was recently reported in B. anthracis suggesting that this pathogen might also regulate density-dependent gene expression via AI-2 (Jones, M. B. and M. J. Blaser. 2003. Infect. Immun. 71:3914-3919). Another protein expressed from a reactive clone in this group was aspartate kinase I (DapG-1), which is involved in the first step of biosynthesis of diaminopimelate from L-aspartate. Diaminopimelate is an important constituent of both the peptidoglycan of vegetative cells and of the spore cortex peptidogylcan of Gram positive bacteria, especially in members of the genus Bacillus. Furthermore, dipicolinate, a by-product during diaminopimelate biosynthesis, is also a part of the spore, comprising as much as 10% of the dry spore weight (Chen, N. Y., et al., 1993. J. Biol. Chem. 268:9448-9465). Aspartokinases play a pivotal role in the biosynthesis of important structural components in diverse microbes.
Several reactive clones expressing proteins involved in the biosynthesis of nucleosides/nucleotides were identified. One such protein was the dihydroorotase, PyrC, which catalyzes one of the reactions in the biosynthesis of uridine monophosphate (UMP) from precursors such as aspartate and glutamine. It is likely that PyrC, as a spore component, functions in pyrimidine nucleotide synthesis during early infection before the elaboration of toxins and other degradative enzymes that cause cellular destruction, and rendering available uracil and other pyrimidine nucleotides to be utilized in the pyrimidine salvage pathway (the closely related B. subtilis possesses a pyrimidine salvage pathway, and hence it is likely that a similar pathway also exists in B. anthracis). PyrC may contribute to B. anthracis survival within the host.
Another protein involved in the synthesis of small molecules was thymidine kinase (Tdk), which functions in pyrimidine salvage (Agrawal, N., et al., 2004. Biochemistry 43:10295-10301). A spore-location alludes to a possible role in salvage of thymidine derivatives from host cells/tissues for DNA synthesis essential for multiplication of B. anthracis following spore-germination. The same cloned insert expressing Tdk also included part of the gene encoding the ribosomal protein L31, which is involved in the synthesis and modification of ribosomal proteins. A clone was also identified expressing the monofunctional, phosphoribosylamine-glycine ligase, PurD, (also called glycinamide ribonucleotide synthetase), an enzyme functioning in de novo purine ribonucleotide biosynthesis. Also in this group was a clone that expressed adenine phosphoribosyltransferase, an enzyme of the purine salvage pathway, which possibly performs a function analogous to the above enzymes of the pyrimidine salvage pathway.
A group of reactive clones expressed proteins involved in the biosynthesis of cofactors, prosthetic groups and carriers. Some expressed proteins functioning in thiamine biosynthesis: ThiC, ThiG, and ThiS. These enzymes function in the de novo synthesis of an important nutrient, namely thiamine (Zhang, Y., et al., 1997. J. Bacteriol. 179:3030-3035; Park, J. H., et al., 2003. Biochemistry 42:12430-12438), suggests a likely role in the in vivo survival of the pathogen. Coupled with the fact that the untranslated regions of mRNA specifying such enzymes contain a metabolite responsive genetic control element or “riboswitch” renders them attractive targets for drug development (Winkler, W., et al., 2002. Nature 419:952-956).
Several clones expressing enzymes involved in protein degradation were identified. One of these was a putative secreted aminopeptidase that belonged to the family of widely distributed metal-associated metalloproteases, which catalyze the removal of N-terminal amino acids from peptides and proteins. The region upstream of the gene encoding this protein has a binding site for PlcR, a pleiotropic regulator of extracellular virulence factors in closely related organisms such as B. thuringiensis (Agaisse, H., et al., 1999. Mol. Microbiol. 32:1043-1053; Read, T. D., et al., 2003. Nature 423:81-86). Although the PlcR homolog in B. anthracis is truncated due to a nonsense mutation, it has been hypothesized that alternative regulatory controls may allow for PlcR-regulated proteins to contribute to B. anthracis virulence (Read, T. D., et al., 2003. Nature 423:81-86). Also, the fact that aminopeptidases are present on the anthrax spore-surface (Liu, H., et al., 2004. J. Bacteriol. 186:164-178), and have been reported to play a role in pathogenesis, particularly of intracellular parasites (Morty, R. E. and J. Morehead. 2002. J. Biol. Chem. 277:26057-26065), suggests that they might play a role in the virulence of B. anthracis. Among other proteins expressed by reactive clones in this group was a peptidase T (PepT-2) (identified twice in this screen), a zinc metalloprotease and an amino tripeptidase, which removes the N-terminal amino acid residue from various tripeptides. Although the contribution of these proteins to the virulence of B. anthracis is unclear, it is of interest that PepT was one of the proteins highly expressed in E. coli K12 biofilms and during growth in preconditioned medium from the laboratory strain E. coli DH5α (Prigent-Combaret, C., et al., 1999. J. Bacteriol. 181:5993-6002), despite the fact that cell-to-cell signaling via acyl homoserine lactone (acyl-HSL) molecules is yet to be demonstrated in Gram positive bacteria, including B. anthracis (Bassler, B. L. 2002. Cell 109:421-424). Another spore-associated protein was a putative prolyl oligopeptidase family protein. Because members, such as dipeptidyl peptidase IV, have been implicated in the virulence of certain bacterial pathogens (Yagishita, H., et al., 2001. Infect. Immun. 69:7159-7161), this protein warrants further study regarding its contribution to the pathogenicity of B. anthracis.
The screen identified two clones expressing regulatory proteins. One of these was a sensory box histidine kinase component of an unknown two-component regulatory system. Although speculative, the fact that sensor kinases sense and transduce signals from the environment to cognate response regulator components to influence gene expression (James A. Hoch and Thomas Silhavy (eds.). 1995. ASM Press, Washington, D.C.), renders it plausible that a spore-surface sensor kinase might be involved in sensing the environment within the macrophage and transducing a signal via its response regulator to affect expression of genes involved in early infection. The other protein identified as part of this group was a LysR-type transcriptional regulator, which in a variety of pathogens is reportedly involved in the positive regulation of diverse classes of genes, including those encoding virulence factors (Schell, M. A. 1993. Annu. Rev. Microbiol. 47:597-626). The screen identified another LysR-type transcriptional regulator encoded on the same insert that also encoded a transporter of the EamA family. The finding that LysR-type regulators were associated with the anthrax spore was not unexpected since such proteins have been identified as constituents of the anthrax spore-surface (Liu, H., et al., 2004. J. Bacteriol. 186:164-178); however, the roles played by these proteins in this location is yet to be defined.
Two reactive clones expressing proteins involved in cellular processes were identified. One of these was an uncharacterized catalase that may be part of the oxidative stress response protecting germinating spores against the lethal effects of H2O2, especially within phagocytic cells. It is plausible that this uncharacterized, spore-associated catalase might act in conjunction with KatX, a catalase present in B. subtilis spores (Bagyan, I., et al., 1998. J. Bacteriol. 180:2057-2062), and with other spore-coat resident enzymes such as superoxide dismutase, to dissipate H2O2 and protect germinating spores against oxidative damage. Other proteins included within this group included a cell division initiation protein, DivIVA, which functions in the proper positioning of the septum during cell-division and also promotes asymmetric septation, an essential prerequisite for sporulation (Cha, J. H. and G. C. Stewart. 1997. J. Bacteriol. 179:1671-1683).
The screen identified a group of clones that expressed proteins of unknown function. Included among these was an acyl transferase of the Gcn5-related acyl transferase (GNAT) superfamily, the members of which are widely distributed in nature and use acyl CoAs to acylate their respective substrates. Interestingly, a paralog in the sequenced B. anthracis Ames strain (BA1085), which is also an acyl transferase of the Gcn5-related acyl transferase (GNAT) superfamily, has been reported to contain the upstream binding motif for the pleiotropic positive regulator of extracellular virulence factor gene expression, PlcR (Read, T. D., et al, 2003. Nature 423:81-86). Also identified by the screen was a carboxyl transferase domain protein, which catalyzes the transfer of a carboxyl group from biotin to an acceptor acyl-CoA, a chlorohydrolase family protein (a family of enzymes that are a large metal dependent hydrolase superfamily); a hydrolase of the carbon-nitrogen hydrolase family functioning in nitrogen metabolism; and an aminotransferase, which catalyzes the transfer of an amino group to a cognate acceptor. Among this group of clones was one that expressed a hydrolase of the alpha/beta fold family with aminopeptidase activity, which was previously reported to be a component of the exosporium of the anthrax spore (Liu, H., et al., 2004. J. Bacteriol. 186:164-178). Also identified was a protein encoded by vrrA (variable region with repetitive sequence), which encodes a 30-kDa protein in the Sterne strain but encodes truncated proteins in the Ames strain and Vollum strain, due to a single nucleotide and a 24-bp deletion, respectively (Andersen, G. L., et al., 1996. J. Bacteriol. 178:377-384). Despite this, the fact the amino acid sequence of VrrA of B. anthracis Sterne differs from that of the closely related B. cereus and B. mycoides at 61 different positions (Andersen, G. L., et al., 1996. J. Bacteriol. 178:377-384), and also the fact that this protein was a target of the AVA-induced immune response in humans suggests that VrrA could be a potential virulence determinant of B. anthracis. Finally, the screen identified a clone expressing a 15.2-kDa hypothetical protein (BA5515) of unknown function. This hydrophilic spore-associated protein was encoded by a 360 bp gene that was present in the sequenced genomes of both B. anthracis Ames and Sterne strains, but not in any of the heretofore-sequenced genomes of close relatives as evidenced by BLAST analysis. Also, no significant homology to other database entries was detected.
In summary, 69 clones expressing anthrax spore-associated proteins targeted by AVA-induced immunity were identified. Positive clones expressed proteins previously identified by other methods as constituents of the anthrax spore-surface, proteins highly expressed during spore germination, proteins that were orthologs of drug targets and virulence determinants of diverse pathogens, and several proteins of unknown function. Of note, the majority of proteins identified by this screen were not spore structural proteins but, rather, proteins expressed during vegetative growth. It is possible that, when on the spore-surface, proteins expressed during vegetative growth-phase that are also spore-associated take on completely different roles than those ascribed to them during vegetative growth, such as those that help establish early infection and spore germination. Such disparate roles for the same protein at different cellular locations have been described previously in other pathogens (Heithoff, D. M., et al, 1999. J. Bacteriol. 181:799-807).
The functions ascribed above to the targets of AVA-induced immunity are putative and not be construed as limiting. More extensive studies to determine the definitive roles of these SA-proteins have commenced. Because the proteins identified in this study are associated with the infective form of Bacillus anthracis (which is likely to interact first with components of the host immune system), and because the expression of a subset of SA-proteins is reportedly increased during spore-germination (Huang, C. M., et al., 2004. Proteomics 4:2653-2661), various approaches are being employed to identify SA-proteins operating during early infection with anthrax spores. Furthermore, because a subset of SA-proteins identified herein were either orthologs of proteins of diverse pathogens under investigation as drug targets, or virulence determinants of both Gram positive and Gram negative bacteria, deletions are being generated of genes encoding selected SA-proteins in various B. anthracis strains to determine the contribution of such proteins to virulence of the pathogen using relevant animal models. The proteins identified by these studies are then further evaluated as an optimally delivered, PA-based vaccine, for protection of appropriate animal models against a challenge with virulent B. anthracis strains. Results of such studies facilitate the development of defined, non-reactogenic anthrax vaccines. In addition, because these proteins are part of the protein repertoire of the spore-surface, a subset of which have been reported to be highly expressed during germination (Huang, C. M., et al., 2004. Proteomics 4:2653-2661), the above-delineated studies help identify SA-proteins with potential for the development of drugs or spore-inactivation strategies. Finally, because of the spore-surface localization of SA-proteins and accessibility to ligands, such as antibodies, experiments have been initiated that are geared toward the identification of B. anthracis-specific domains within SA-proteins, as well as toward confirmation of spore-surface-localization of such domains for the development of assays for spore-detection.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications can be practiced. Therefore, the description and examples should not be construed as limiting the scope of the invention, which is delineated by the appended numbered claims.
This example will prophetically describe the further evaluation of identified anthrax spore associated proteins.
Identification of individual spore-associated proteins that contribute to protective immunity, and optimization of formulation and delivery of such proteins to the immune system, can result in the development of more efficacious second and third generation multivalent anthrax vaccines. Because protective efficacy of a multivalent vaccine comprising these proteins cannot be directly studied in humans due to ethical reasons, the vaccine potential of these proteins can be addressed in animal models (in accordance with the recent “animal rule” see Food and Drug Administration “New drug and biological drug products: evidence needed to demonstrate effectiveness of new drugs when human efficacy studies are not ethical or feasible.” Fed Regist 2002; 67:37988-98] criteria proposed by the US Food and Drug Administration [FDA] for demonstrating vaccine effectiveness in situations that preclude human volunteer challenge studies by allowing reasonably well-understood models to substitute for human studies).
The following scheme for further evaluation of the vaccine potential of identified proteins will be accomplished using well-established experimental methods:
Step 1. Each protein will be purified to homogeneity/near homogeneity using defined sequential chromatographic protein purification techniques. Proteins that are difficult to purify will be subjected to bioinformatics to select hydrophilic, surface-exposed domains (most likely to be recognized by the immune system), which will then be chemically synthesized.
Step 2. Step 1 will be followed by a preliminary evaluation of the vaccine potential of each protein using A/J mice (a mouse strain that is highly susceptible to the attenuated, experimental Bacillus anthracis Sterne strain which can be used in these experiments). Defined amounts of each purified protein will be injected intraperitoneally into groups of A/J mice without and with appropriate adjuvants on day 0, and boosted again on day 14. Immunized mice will then be challenged on day 28 with a defined number B. anthracis Sterne (the anthrax vaccine approved for human-use in the USA is derived from the culture supernatant of a related strain) with 10×LD50 spores via intranasal instillation or aerosol. The “time to death” will be noted for each group and compared with that for unimmunized mice, and survival curves will be plotted. Spore-associated proteins that significantly increases the time to death/completely protect mice are vaccine candidates that warrant further study.
Step 3. The next batch of experiments will involve the evaluation of the vaccine candidate proteins as a multivalent experimental vaccine administered via transcutaneous immunization (TCI). Transcutaneous immunization [TCI] is a needle-free method of immunization that involves application of protein antigens co-administered with an adjuvant on intact skin, resulting in the development of robust systemic and mucosal immune responses both against the antigens, as well as the adjuvant. For TCI, vaccine candidate proteins will be pooled, and used to immunize A/J mice transcutaneously along with cholera toxin (CT) as an adjuvant. The experimental vaccine will contain 50 μg of each protein will be administered with 50 μg of adjuvant and without or with 50 μg of protective antigen (PA), the nontoxic receptor binding moiety of anthrax toxins, which is the principal component of AVA. These experiments will allow a direct comparison and evaluation of the efficacy of the experimental vaccine with and without PA, and hence might dictate the use of the multivalent experimental vaccine either as a more efficacious second generation (with PA) or a novel third generation anthrax vaccine (without PA). Several groups of A/J mice will be administered a primary immunization (day 0) or a primary and a booster immunization (day 0 and day 14) via TCI with the respective experimental vaccine formulation. Mice will then be challenged as described above on day 28. Efficacy will be assessed by the ability of the experimental vaccine to protect A/J mice against a lethal challenge with B. anthracis, compared with that of a control group of unimmunized A/J mice. The duration of protective immunity will then be assessed by challenging A/J mice at 28-day intervals (starting from day 28 to day 168).
Step 4. A parallel set of identical experiments will be performed using CpG oligonucleotides as an adjuvant instead of CT.
Step 5. The above set of experiments should yield information leading to optimization of the immunization regimen, formulation of the multivalent experimental vaccine and the best adjuvant for induction of long-lasting protective immunity. To confirm efficacy, the multivalent experimental vaccine will evaluated in another mammalian species, namely, rabbits, via TCI using the identical experimental protocol described above. Immunized rabbits will be challenged as outlined above using fully virulent strains of B. anthracis.
Step 6. Similar experiments will also be performed in both mice and rabbits, in which genes encoding spore-associated proteins will be cloned into suitable plasmid DNA vectors and administered as a multivalent genetic (DNA) vaccine.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis str. Sterne, complete genome.
Bacillus anthracis strain A16R protective antigen (pag) gene, complete cds.
Bacillus anthracis strain A16R protective antigen (pag) gene, complete cds.
Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.
This application claims the benefit of U.S. Provisional Application No. 60/700,645, filed Jul. 19, 2005, the entire contents of which are expressly incorporated herein by reference. Each of the applications and patents cited in this text, as well as each document or reference cited in each of the applications and patents (including during the prosecution of each issued patent; “application cited documents”), and each of the PCT and foreign applications or patents corresponding to and/or paragraphing priority from any of these applications and patents, and each of the documents cited or referenced in each of the application cited documents, are hereby expressly incorporated herein by reference. More generally, documents or references are cited in this text, either in a Reference List before the paragraphs, or in the text itself; and, each of these documents or references (“herein-cited references”), as well as each document or reference cited in each of the herein-cited references (including any manufacturer's specifications, instructions, etc.), is hereby expressly incorporated herein by reference.
The United States government may have certain rights in this invention by virtue of grant number R21 AI055968-01 from the National Institutes of Health.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US06/28015 | 7/19/2006 | WO | 00 | 4/30/2009 |
| Number | Date | Country | |
|---|---|---|---|
| 60700645 | Jul 2005 | US |
