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WO2007011411A2 - Fragment n du facteur oedematogene utilise comme antigene pour l'immunisation contre l'anthrax - Google Patents

Fragment n du facteur oedematogene utilise comme antigene pour l'immunisation contre l'anthrax Download PDF

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Publication number
WO2007011411A2
WO2007011411A2 PCT/US2005/039799 US2005039799W WO2007011411A2 WO 2007011411 A2 WO2007011411 A2 WO 2007011411A2 US 2005039799 W US2005039799 W US 2005039799W WO 2007011411 A2 WO2007011411 A2 WO 2007011411A2
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nucleic acid
polypeptide
disclosed
antigen
anthrax
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WO2007011411A3 (fr
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Michael E. Pichichero
Mingtao Zeng
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University of Rochester
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University of Rochester
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/07—Bacillus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination

Definitions

  • Figure 1 shows validation of adenoviral vector encoding EFn by PCR.
  • Lane 1 DNA marker, ⁇ -DNA digested with HinaTE;
  • Lane 2 plasmid pBHGlOX/delta El, E3Cre as DNA template in PCR (positive control for Ad5 Fiber);
  • Lane 4-8 Genomic DNA of adenovirus Ad/EFn.
  • the primers used in the PCR are indicated in the figure.
  • polypeptide can comprise amino acids 1-254 of SEQ ID NO:1 or it can comprise the amino acid sequence of SEQ ID NO: 3.
  • nucleic acids encoding anu of the disclosed amino acid sequences, as discussed herein, such as SEQ ID NO:4.
  • nucleic acids for example, comprising a sequence having at least 80% homology to SEQ ID NO: 4 or a nucleic acid comprising 15 or more nucleotides with at least 90% homology to SEQ ID NO: 4.
  • cells comprising any of the polypeptides disclosed herein, nucleic acids disclosed herein, or vectors disclosed herein, or fragments thereof.
  • vaccines comprising one or more of the polypeptides disclosed herein or nucleic acids disclosed or fragment thereof.
  • combination vaccines comprising purified antigen, wherein the antigen comprises any one of the polypeptides or nucleic acids disclosed herein, or fragment thereof, for example, related to EF, and a polypeptide or nucleic acid, or fragment thereof, comprising PA, LF.
  • the immune response is a ThI or Th2 immune response.
  • anthracis antigens can contribute in a significant manner to protective immunity (Brassier, F., and M. Mock. 2001. Toxicon 39:1747-55) (Cohen, S et al. 2000. Infect hnmun 68:4549-58).
  • PA is responsible for the internalization of LF and EF in the the cytosol of eukaryotic cells where they act as metalloproteases and adenylylcyclases, respectively, to disrupt cell activity.
  • LF and EF have extensive sequence homology in residues 1-300, and it is these regions that are responsible for high affinity binding to PA. Residues
  • fragments of EF such as polypeptides comprising the N-terminal domain, which is the non-enzymatic region of EF (EFn).
  • EFn non-enzymatic region of EF
  • these N-terminal fragments of EF can be useful as a non- toxic antigen for immunization against anthrax.
  • the use of the disclosed fragments as an antigen can generate an immune response to both EF and LF toxins.
  • EF antigens that are N-terminal fragments of EF comprising amino acids 1-254 of SEQ ID NO:1, or molecules having at least 70, 80, 90, 95% homology, or as discussed herein, to SEQ ID NO:1 or 3.
  • EF antigens that are fragments of EF with the sequence of SEQ ID NO:3.
  • EF antigens that are variants of SEQ ID NO:3 with conservative amino acid substitutions.
  • nucleic acids that encode any of the EF antigens disclosed and a vector containing these nucleic acids for expression and delivery.
  • the vector is a replication-incompetent adenoviral plasmid (pBHGlox(delta)El,E3Cre) containing nucleotides 1-762 of the cya gene SEQ ID NO:4, which encodes for amino acids 1-254 of the mature EF protein SEQ ID NO:3.
  • the final adenovirus expresses the 1-254 amino acids of EFn plus an additional 68 amino acid tag.
  • AU disclosed polypeptide fragments and variants of EF and the nucleic acids that encode the said polypeptides can be used as antigens in the production of antibodies or as vaccines for immunity against anthrax infection.
  • compositions designed to specifically bind or induce binding to the said EF variants and fragments, or other antigens as discussed herein, in combination with EF, such as LF or PA include purified antibodies and nucleic acids that specifically target non- native EF variants and fragments as well as the methods to illicit an immune response to EF using EF fragments and variants. It is understood that the discussion for molecules targeting EF or EF fragments is also applicable to PA or LF or any other antigen disclosed herein, and for example, could be used in combination with EF antigen.
  • the antibodies can also be encapsulated, for example into lipsomes, microspheres, or other transfection enhancement agents, for improved delivery into the cells to maximize the treatment efficiency.
  • the gene sequences encoding the provided antibodies, or their fragments such as Fab fragments can further be cloned into genetic vectors, such as plasmid or viral vectors, for example, and delivered into the hosts for endogenouse expression of the antibodies for treatment of anthrax.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature, 321 :522- 525 (1986); Riechmami et al., Nature, 332:323-327 (1988); and Presta, Curr. Op. Struct. Biol, 2:593-596 (1992)).
  • Fc immunoglobulin constant region
  • More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the SaIk Institute Cell Distribution Center, San Diego, Calif, and the American Type Culture Collection, Rockville, Md. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., "Monoclonal Antibody Production Techniques and Applications” Marcel Dekker, Inc., New York, (1987) pp. 51-63). The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against an EF fragment, as disclosed herein.
  • the Fab fragments produced in the antibody digestion also contain the constant domains of the light chain and the first constant domain of the heavy chain.
  • Functional nucleic acid molecules can interact with any macromolecule, such as DNA, RNA, polypeptides, or carbohydrate chains.
  • functional nucleic acids can interact with the niRNA of the disclosed EF fragments or the genomic DNA of the disclosed EF fragments or they can interact with the EF fragments.
  • Often functional nucleic acids are designed to interact with other nucleic acids based on sequence homology between the target molecule and the functional nucleic acid molecule, hi other situations, the specific recognition between the functional nucleic acid molecule and the target molecule is not based on sequence homology between the functional nucleic acid molecule and the target molecule, but rather is based on the formation of tertiary structure that allows specific recognition to take place.
  • Antisense molecules are designed to interact with a target nucleic acid molecule through either canonical or non-canonical base pairing. The interaction of the antisense molecule and the target molecule is designed to promote the destruction of the target molecule through, for example, RNAseH mediated RNA-DNA hybrid degradation. Alternatively the antisense molecule is designed to interrupt a processing function that normally would take place on the target molecule, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule. Numerous methods for optimization of antisense efficiency by finding the most accessible regions of the target molecule exist. Exemplary methods would be in vitro selection experiments and DNA modification studies using DMS and DEPC.
  • Ribozymes are nucleic acid molecules that are capable of catalyzing a chemical reaction, either intramolecularly or intermolecularly. Ribozymes are thus catalytic nucleic acid. It is preferred that the ribozymes catalyze intermolecular reactions.
  • EF and LF toxins include single, double, triple and multiple immunizations.
  • One skilled in the art would be able to determine the effectiveness of EF variants and fragments as immunogens by evaluating serum antibodies to EF and LF in immunized subjects.
  • Successful immunization can result in anti-EF and anti-LF IgG titers, for example, of at least 100, 400, andl600 following 1, 2, and 3 immunizations, respectively, 12 weeks post-immunization, in a mouse for example.
  • the EF polypeptide fragments discussed herein can be used in the construction of a vaccine comprising an immunogenic amount of the antigen and a pharmaceutically acceptable carrier.
  • the vaccine can be an antigen of the present invention or the antigen bound to a carrier or a mixture of bound or unbound antigens.
  • the vaccine can then be used in a method of preventing anthrax infection.
  • Immunogenic amounts of the antigen can be determined using standard procedures. Briefly, various concentrations of a putative specific immunoreactive peptides or polypeptides are prepared, administered to an animal, such as a human, and the immunological response (e.g., the production of antibodies or cell-mediated response) of an animal to each concentration is determined.
  • the pharmaceutically acceptable carrier in the vaccine can comprise saline or other suitable carriers (Arnon, R. (Ed.) Synthetic Vaccines 1:83-92, CRC Press, Inc., Boca Raton, Florida, 1987).
  • An adjuvant can also be a part of the carrier of the vaccine, in which case it can be selected by standard criteria based on the antigen used, the mode of administration and the subject (Arnon, R. (Ed.), 1987).
  • Methods of administration can be by oral or sublingual means, or by injection, depending on the particular vaccine used and the subject to whom it is administered.
  • Protein variants and derivatives are well understood to those of skill in the art and in can involve amino acid sequence modifications. For example, amino acid sequence modifications typically fall into one or more of four classes: substitutional, insertional, truncational or deletional variants. Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues.
  • Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues.
  • Immunogenic fusion protein derivatives are made by fusing a polypeptide sufficiently large to confer immunogenicity to the target sequence by cross-linking in vitro or by recombinant cell culture transformed with DNA encoding the fusion.
  • Truncations are characterized by the removal of amino acids from the C-terminus or N-terminus of the full length protein. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 2 to 6 residues are deleted at any one site within the protein molecule.
  • substitutions may be combined to arrive at a final construct.
  • the mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary rnRNA structure.
  • substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Tables 1 and 2 and are referred to as conservative substitutions.
  • Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table 2, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain.
  • substitutions which in general are expected to produce the greatest changes in the protein properties will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g.
  • Certain post-translational derivatizations are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and asparyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the o-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco pp 79-86 [1983]), acetylation of the N-terminal amine and, in some instances, amidation of the C-terminal carboxyl.
  • Molecules can be produced that resemble peptides, but which are not connected via a natural peptide linkage.
  • a particularly preferred non-peptide linkage is -CH 2 NH-. It is understood that peptide analogs can have more than one atom between the bond atoms, such as b-alanine, g- aminobutyric acid, and the like.
  • Amino acid analogs and analogs and peptide analogs often have enhanced or desirable properties, such as, more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others.
  • the phosphate moiety of a nucleotide is pentavalent phosphate.
  • An non-limiting example of a nucleotide would be 3'-AMP (3 '-adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate).
  • Conjugates can be chemically linked to the nucleotide or nucleotide analogs. Such conjugates include but are not limited to lipid moieties such as a cholesterol moiety. (Letsinger et al, Proc. Natl. Acad. Sci. USA, 1989,86, 6553-6556), 119.
  • a Watson-Crick interaction is at least one interaction with the Watson-
  • the vector carrying the DNA of choice When the vector carrying the DNA of choice is transfected into these cell lines, the vector containing the gene of interest is replicated and packaged into new retroviral particles, by the machinery provided in cis by the helper cell. The genomes for the machinery are not packaged because they lack the necessary signals.
  • Non-nucleic acid based systems include, for example, replicating and host- restricted non-replicating vaccinia virus vectors.
  • compositions can be delivered to the target cells in a variety of ways.
  • the compositions can be delivered through electroporation, or through lipofection, or through calcium phosphate precipitation.
  • the delivery mechanism chosen will depend in part on the type of cell targeted and whether the delivery is occurring for example in vivo or in vitro.
  • the compound can be administered as a component of a microcapsule that can be targeted to specific cell types, such as macrophages, or where the diffusion of the compound or delivery of the compound from the microcapsule is designed for a specific rate or dosage.
  • Expression systems 151 The nucleic acids that are delivered to cells typically contain expression controlling systems.
  • the inserted genes in viral and retroviral systems usually contain promoters, and/or enhancers to help control the expression of the desired gene product.
  • a promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site.
  • a promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements.
  • Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. beta actin promoter.
  • viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. beta actin promoter.
  • the early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication (Fiers et al, Nature, 273: 113 (1978)).
  • Enhancers function to increase transcription from nearby promoters. Enhancers also often contain response elements that mediate the regulation of transcription. Promoters can also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression of a gene. While many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, -fetoprotein and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression.
  • Preferred examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. 154.
  • the promotor and/or enhancer may be specifically activated either by light or specific chemical events which trigger their function.
  • Systems can be regulated by reagents such as tetracycline and dexamethasone.
  • the promoter and/or enhancer region can act as a constitutive promoter and/or enhancer to maximize expression of the region of the transcription unit to be transcribed.
  • the promoter and/or enhancer region be active in all eukaryotic cell types, even if it is only expressed in a particular type of cell at a particular time.
  • a preferred promoter of this type is the CMV promoter (650 bases).
  • Other preferred promoters are SV40 promoters, cytomegalovirus (full length promoter), and retroviral vector LTF.
  • GFAP glial fibrillary acetic protein
  • Expression vectors used in eukaryotic host cells may also contain sequences necessary for the termination of transcription which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3' untranslated regions also include transcription termination sites. It is preferred that the transcription unit also contain a polyadenylation region. One benefit of this region is that it increases the likelihood that the transcribed unit will be processed and transported like mRNA.
  • the identification and use of polyadenylation signals in expression constructs is well established. It is preferred that homologous polyadenylation signals be used in the transgene constructs.
  • the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of about 400 bases. It is also preferred that the transcribed units contain other standard sequences alone or in combination with the above sequences improve expression from, or stability of, the construct.
  • These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genes necessary for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media.
  • An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements. Individual cells which were not transformed with the DHFR or TK gene will not be capable of survival in non-supplemented media.
  • the antibody level following the first exposure to a vaccine antigen referred to as primary antibody response consists primarily of IgM, is of brief duration, and low intensity, i.e., inadequate for effective protection.
  • the antibody level following the second and subsequent antigenic challenges, or secondary antibody response appears more quickly and persists for a longer period, attains a higher titer, and consists predominantly of IgG.
  • the shorter latent period is due to antigen-sensitive cells, called memory cells, already present at the time of repeat exposure.
  • the adenovirus vectored vaccine could be administrated by different routes to achieve immunization.
  • the current anthrax vaccine can function with 6 immunizations over a period of 18 months followed by annual boosters.
  • the disclosed vaccines can work with 1, 2, 3, 4, or 5 immunizations to provide protective immunity with optional boosters.
  • the third immunization can be given 8 weeks and result in anti-EF IgGl, IgG2a, and IgG antibody titers of 1600-6400, 800-3200, and 1600-6400, respectively in week 12 after primary immunization.
  • Antibody titers are defined as the highest dilution in post-immune sera that resulted in equal absorbance value of pre-immune samples for each subject. 167.
  • the vaccines disclosed herein provide anti-EF and anti-LF IgG antibody titers of 100, 200, 300, 400, 500, 600, 700, 800, 900, 100O 5 1500, 2000, 2500, 3000, 3500,.4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 10500, 11000, 11500, and 12000, and titers of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 10500, 11000, 11500, and 12000, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 weeks post-immunization following 1, 2, 3, 4, 5, or more immunizations.
  • the vaccines disclosed herein provide anti-EF and anti-LF IgGl antibody titers of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 10500, 11000, 11500, and 12000, and titers of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 10500, 11000, 11500, and 12000, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 weeks post-immunization following 1, 2, 3, 4, 5, or more immunizations.
  • the vaccines disclosed herein provide anti-EF and anti-LF IgG2a antibody titers of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 10500, 11000, 11500, and 12000, and titers of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 10500, 11000, 11500, and 12000, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 weeks post-immunization following 1, 2, 3, 4, 5, or more immunizations .
  • Boosters can be given every 1, 2, 3, 4, 6, 8, 12 years following prior inoculation, for example.
  • compositions 171 Methods of making the compositions 171.
  • the compositions disclosed herein and the compositions necessary to perform the disclosed methods can be made using any method known to those of skill in the art for that particular reagent or compound unless otherwise specifically noted. It is also understood that basic recombinant biotechnology methods can be used to produce the nucleic acids and proteins disclosed herein. 1. Nucleic acid synthesis
  • the nucleic acids such as, the oligonucleotides to be used as primers can be made using standard chemical synthesis methods or can be produced using enzymatic methods or any other known method. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see for example, Sambrook et ah, Molecular Cloning: A Laboratory Manual, 2nd Edition
  • a peptide or polypeptide corresponding to the disclosed proteins can be synthesized by standard chemical reactions.
  • a peptide or polypeptide can be synthesized by standard chemical reactions.
  • a peptide or polypeptide can be synthesized by standard chemical reactions.
  • a peptide or polypeptide can be synthesized by standard chemical reactions.
  • a peptide or polypeptide can be synthesized by standard chemical reactions.
  • a peptide or polypeptide can be synthesized by standard chemical reactions.
  • a peptide or polypeptide can be synthesized by standard chemical reactions.
  • a peptide or polypeptide can be synthesized by standard chemical reactions.
  • a peptide or polypeptide can be synthesized by standard chemical reactions.
  • a peptide or polypeptide can be synthesized by standard chemical reactions.
  • a peptide or polypeptide can be synthesized by standard chemical reactions.
  • enzymatic ligation of cloned or synthetic peptide segments allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains (Abrahmsen L et al., Biochemistry, 30:4151 (1991)).
  • native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two step chemical reaction (Dawson et al. Synthesis of Proteins by Native Chemical Ligation. Science, 266:776-779 (1994)).
  • nucleic acids in SEQ E) NOs: 4-10 and 12-13 are disclosed.
  • methods that can be used for making these compositions such as synthetic chemical methods and standard molecular biology methods. It is understood that the methods of making these and the other disclosed compositions are specifically disclosed.
  • nucleic acid molecules produced by the process comprising linking in an operative way a nucleic acid comprising the sequence set forth in SEQ ID NO:4 and a sequence controlling the expression of the nucleic acid.
  • nucleic acid molecules produced by the process comprising linking in an operative way a nucleic acid molecule comprising a sequence having 80% identity to a sequence set forth in SEQ ID NO:4, and a sequence controlling the expression of the nucleic acid.
  • nucleic acid molecules produced by the process comprising linking in an operative way a nucleic acid molecule comprising a sequence encoding a peptide having 80% identity to a peptide set forth in SEQ ID NO:3 and a sequence controlling an expression of the nucleic acid molecule.
  • nucleic acids produced by the process comprising linking in an operative way a nucleic acid molecule comprising a sequence encoding a peptide having 80% identity to a peptide set forth in SEQ ID NO:3, wherein any change from the sequence are conservative changes, and a sequence controlling an expression of the nucleic acid molecule.
  • animals produced by the process of transfecting a cell within the animal with any of the nucleic acid molecules disclosed herein Disclosed are animals produced by the process of transfecting a cell within the animal any of the nucleic acid molecules disclosed herein, wherein the animal is a mammal. Also disclosed are animals produced by the process of transfecting a cell within the animal any of the nucleic acid molecules disclosed herein, wherein the mammal is mouse, rat, rabbit, cow, sheep, pig, or primate.
  • Example 1 N-Fragment of Edema Factor as a New Candidate Antigen for Immunization against Anthrax Using Replication- Incompetent Adenovirus as a Delivery Vector
  • the nontoxic N-terminal fragment of Bacillus anthracis edema factor (EF) as a candidate antigen in an anthrax vaccine was evaluated, using a replication-incompetent adenoviral vector.
  • An E1/E3 deleted adenovirus Ad/EFn encoding the N-terminal region 1-254 amino acids SEQ ID NO:3 of the edema factor (EFn) with a 68 amino acids tag SEQ ID NO: 7 was constructed using the native DNA sequence of EFn SEQ ID NO:4.
  • the positive PCR results indicated that the EFn was cloned into the adenoviral vector with the correct orientation downstream of the mCMV promoter.
  • the unique 1.7 kb Ad5 fiber gene fragment in the adenovirus backbone was co-amplified by PCR with the primers 5Fb/SEQ ID NO:9 (5'-CCGTCTGAAGATACCTTCAA-S') and 3Fb/ SEQ ID NO:10 (5'-ACCAGTCCCATGAAAATGAC-S'), indicating the correct recombinant adenovirus Ad/EFn was constructed.
  • PCR products were fractionated in a 1% agarose gel, stained with ethidium bromide, and visualized with the Kodak Imaging System 440CF (Fig. 1).
  • the final adenovirus Ad/EFn expresses the 1-254 amino acids of EFn plus the 68 amino acids tag under the transcriptional control of mCMV early promoter.
  • mice Four to six week old female A/ J mice were purchased from Jackson Laboratory (Bar harbor, ME), allotted into treatment and control groups (4 mice/cage), and housed under BSL2 pathogen-free conditions in the animal facility at University of Rochester. The mice received intramuscular injections into the hind-leg quadriceps with 1 x 10 8 pfu/dose adenovirus Ad/EFn in treatment groups or an equal volume of buffer (10 mM Tris pH7.5, 135 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , and 1 M sucrose) in control groups. Animals were immunized with Ad/EFn one time (week 0), two times (week 0, 4), or three times (week 0, 4, 8). Animal sera were collected every two weeks by retro-orbital bleeding and stored at -2O 0 C until assayed. The animal research herein reported was conducted in facilities with programs accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care.
  • mice sera in PBS containing 0.05% Tween 20 100 ⁇ l serial dilutions of mice sera in PBS containing 0.05% Tween 20 were added to each well (starting with 1:100 dilution), and incubated for 2 hrs at room temperature. The plates were then washed with washing buffer 5 times and incubated with 100 ⁇ l/well 1:10000 dilution of goat anti-mouse IgG, IgGl or IgG2a conjugated to horseradish peroxidase (Calbiochem, CA ) for 1 hr at room temperature.
  • Edtx (EF + PA) is able to increase the intracellular levels of cyclic AMP (cAMP) in Chinese hamster ovary (CHO) cells (Leppla, 1982). Neutralizing antibodies to EF were measured by the ability of sera to neutralize this cytotoxicity of Edtx in CHO cells.
  • the CHO-Kl cells (ATTC #CCL-61) were seeded in a 96-well tissue culture plate at a density of 40,000 cells/well in 100 ⁇ l Khaigan's modified F- 12 medium containing 10% fetal bovine serum, and incubated at 37 0 C, 5% CO 2 for 24 h as previously described (Zmuda, 2005).
  • OD optical density
  • LF protein List Biological Laboratories, CA
  • PA protein List Biological Laboratories, CA; at 60 ng/ml final concentration
  • Fig. 4A shows that although one or two vaccine doses could not protect animals against the challenge with 20OxLD 50 of B. anthracis Sterne spores, immunization significantly changed the survival distribution in the four groups (P-value 0.0062).
  • the mean survival time was 6.4 days in the control group and 6.9, 7.3 and 8.1 days for one, two and three immunization groups, respectively. Pairwise comparison showed that three immunizations with Ad/EFn significantly changed the survival when compared to the control group (P-value 0.0071). This indicated that booster immunizations enhanced the protective immunity in animals.
  • Fig. 4B shows that the survival rate of the immunized animals is highly dependent on the dosage of spore used in the challenge experiments. After animals were immunized three times with Ad/EFn, 57% could survive for 2 weeks from a 10OxLD 50 spore challenge; however, only 37% survived from 20OxLD 50 spore challenge.
  • Bacillus anthracis protective antigen expressed in Salmonella typhimurium SL 3261, affords protection against anthrax spore challenge.
  • Anthrax toxin edema factor abacterial adenylate cyclase that increases cyclic AMP concentrations of eukaryotic cells.
  • Lukashok SA Horwitz MS. New perspectives in adenoviruses. Curr Clin Topics Infect Dis 1998;18:286-305.

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  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Genetics & Genomics (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne des compositions et des procédés permettant d'obtenir un fragment non catalytique du facteur oedématogène de Bacillus anthracis. En particulier, l'invention porte sur un peptide ou un acide nucléique codant pour un peptide comprenant un fragment non catalytique du facteur oedématogène. L'invention se rapporte en outre à des procédés permettant d'utiliser ce fragment comme antigène de l'anthrax afin de déclencher une réponse immunitaire efficace pour traiter ou prévenir l'infection par l'anthrax.
PCT/US2005/039799 2004-11-05 2005-11-04 Fragment n du facteur oedematogene utilise comme antigene pour l'immunisation contre l'anthrax Ceased WO2007011411A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US62538204P 2004-11-05 2004-11-05
US60/625,382 2004-11-05

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WO2007011411A2 true WO2007011411A2 (fr) 2007-01-25
WO2007011411A3 WO2007011411A3 (fr) 2011-04-21

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PCT/US2005/039799 Ceased WO2007011411A2 (fr) 2004-11-05 2005-11-04 Fragment n du facteur oedematogene utilise comme antigene pour l'immunisation contre l'anthrax

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009063507A1 (fr) * 2007-11-12 2009-05-22 Rakesh Bhatnagar Vaccin d'adn contre le charbon

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6329156B1 (en) * 1999-03-22 2001-12-11 The Regents Of The University Of California Method for screening inhibitors of the toxicity of Bacillus anthracis

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009063507A1 (fr) * 2007-11-12 2009-05-22 Rakesh Bhatnagar Vaccin d'adn contre le charbon

Also Published As

Publication number Publication date
WO2007011411A3 (fr) 2011-04-21

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