Patent application title: METHODS AND MATERIALS FOR PRODUCING IMMUNE RESPONSES AGAINST POLYPEPTIDES INVOLVED IN ANTIBIOTIC RESISTANCE
Inventors:
Michael A. Barry (Rochester, MN, US)
IPC8 Class: AA61K39085FI
USPC Class:
4241901
Class name: Antigen, epitope, or other immunospecific immunoeffector (e.g., immunospecific vaccine, immunospecific stimulator of cell-mediated immunity, immunospecific tolerogen, immunospecific immunosuppressor, etc.) amino acid sequence disclosed in whole or in part; or conjugate, complex, or fusion protein or fusion polypeptide including the same disclosed amino acid sequence derived from bacterium (e.g., mycoplasma, anaplasma, etc.)
Publication date: 2011-08-04
Patent application number: 20110189217
Abstract:
This document relates to methods and materials for producing immune
responses against polypeptides involved in antibiotic resistance. For
example, vaccines against polypeptides involved in antibiotic resistance
as well as methods for vaccinating mammals against polypeptides involved
in antibiotic resistance are provided.Claims:
1. A method for inducing an immune response against a polypeptide
involved in antibiotic resistance, said method comprising administering
to an animal an amount of said polypeptide or a nucleic acid encoding
said polypeptide effective for producing said immune response.
2. The method of claim 1, wherein said polypeptide is a blaZ polypeptide, a mecA polypeptide, a whiB7 polypeptide, a tap polypeptide, a RV1473 polypeptide, a katG polypeptide, an inhA polypeptide, a rpoB polypeptide, a gidB polypeptide, a pncA polypeptide, an embB polypeptide, or a gyrA polypeptide.
3. The method of claim 1, wherein said polypeptide is a blaZ polypeptide.
4. The method of claim 1, wherein said polypeptide is a mecA polypeptide.
5. The method of claim 1, wherein said antibiotic resistance is penicillin-resistance.
6. The method of claim 1, wherein said antibiotic resistance is methicillin-resistance.
7. The method of claim 1, wherein said antibiotic resistance is vancomycin-resistance.
8. The method of claim 1, wherein said animal is a human.
9. The method of claim 1, wherein said method comprises administering said polypeptide to said animal.
10. The method of claim 1, wherein said method comprises administering said nucleic acid to said animal.
11. The method of claim 10, wherein said nucleic acid is a viral vector encoding said polypeptide.
12. The method of claim 11, wherein said viral vector is an adenoviral, vaccinia viral, measles, or adeno-associated virus vector.
13. The method of claim 1, wherein said immune response reduces the severity of an infection within said animal.
14. The method of claim 1, wherein said infection is a Mycobacterium tuberculosis infection.
15. The method of claim 1, wherein said infection is a Staphylococcus aureus infection.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of priority from U.S. Provisional Application Ser. No. 61/075,881, filed on Jun. 26, 2008.
BACKGROUND
[0002] 1. Technical Field
[0003] This document relates to methods and materials for producing immune responses against polypeptides involved in antibiotic resistance. For example, this document provides vaccines against polypeptides involved in antibiotic resistance as well as methods for vaccinating mammals against polypeptides involved in antibiotic resistance.
[0004] 2. Background Information
[0005] For many pathogens, reasonably effective antibiotics and vaccines that can temper or control infections, symptoms, and fatalities are available. However, when pathogens become resistant to effective antibiotics, these normally controlled pathogens can become lethal for the patient and can amplify into to epidemics with no effective treatment. Tuberculosis is an example of this problem. Mycobacterium tuberculosis (Mtb) can be effectively treated with triple antibiotic therapies. However, use of antibiotics has selected Mtb that is either multi-drug resistant (MDR) or extensively drug resistant (XDR), both of which are substantially more difficult to treat with multi-drug therapy. MDR and XDR Mtb have acquired drug resistance by accumulating multiple gene products that can inactivate or efflux multiple antibiotics.
SUMMARY
[0006] This document relates to methods and materials for producing immune responses against polypeptides involved in antibiotic resistance. For example, this document provides vaccines against polypeptides involved in antibiotic resistance as well as methods for vaccinating mammals against polypeptides involved in antibiotic resistance.
[0007] In general, one aspect of this document features a method for inducing an immune response against a polypeptide involved in antibiotic resistance. The method comprises, or consists essentially of, administering to an animal (e.g., a mammal) an amount of the polypeptide or a nucleic acid encoding the polypeptide effective for producing the immune response. The polypeptide can be a blaZ polypeptide, a mecA polypeptide, a whiB7 polypeptide, a tap polypeptide, a RV1473 polypeptide, a katG polypeptide, an inhA polypeptide, a rpoB polypeptide, a gidB polypeptide, a pncA polypeptide, an embB polypeptide, or a gyrA polypeptide. The antibiotic resistance can be penicillin-resistance. The antibiotic resistance can be methicillin-resistance. The antibiotic resistance can be vancomycin-resistance. The animal can be a human. The method can comprise administering the polypeptide to the animal. The method can comprise administering the nucleic acid to the animal. The nucleic acid can be a viral vector encoding the polypeptide. The viral vector can be an adenoviral, vaccinia viral, measles, or adeno-associated virus vector. The immune response can reduce the severity of an infection within said animal. The infection can be a Mycobacterium tuberculosis infection. The infection can be a Staphylococcus aureus infection.
[0008] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
[0009] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 is a photograph of a western blot of cell lysates obtained from mammalian cells that were transfected with plasmids expressing either a BlaZ polypeptide or a MecA polypeptide. Proteins were detected with a anti-His6 antibody recognizing a His6 tag on the c-terminus of each of the proteins. The left band are normal cells. The right band of each set are cells expressing Rad23 that inhibits proteasome degradation of proteins. The negative control (-ve control) is a lysate of cells transfected with GFP gene.
[0011] FIG. 2 is photograph of Western blot of BlaZ polypeptides purified from a cell lysate and from the media of bacterial cells. CBD designates the cellulose binding domain fusion tag of the pET38 vector. BlaZ-CBD is the fusion of BlaZ to the CBD tag.
DETAILED DESCRIPTION
[0012] This document provides methods and materials for producing immune responses against polypeptides involved in antibiotic resistance. For example, this document provides vaccines against polypeptides involved in antibiotic resistance as well as methods for vaccinating mammals against polypeptides involved in antibiotic resistance.
[0013] The vaccines provided herein can be in the form of recombinant polypeptides involved in antibiotic resistance or nucleic acid vectors (e.g., viral vectors) designed to express such recombinant polypeptides. The vaccines provided herein can be used to immunize or treat any type of animal including, without limitation, humans, cows, pigs, poultry, dogs, and cats. The vaccines provided herein can be used to induce an immune response against any type of pathogen including, without limitation, intracellular pathogens such as mycobacterium, chlamydia, rickettsiae, lysteria, and brucella as well as extracellular pathogens such as staphylococci, streptococci, enterococci, pneumococci, gram positive bacteria, and anthrax. For example, the vaccines provided herein can be designed to target β-lactamase and penicillin binding proteins to treat or prevent community and hospital-acquired bacterial infections such as methicillin-resistant Staphylococcus aureus.
[0014] Examples of polypeptides involved in antibiotic resistance include, without limitation, a blaZ polypeptide (see Entrez Gene ID: 2859819) from S. aureus; a mecA polypeptide (Entrez Gene ID: 2861157) from S. aureus; a whiB7 polypeptide from Mtb (Entrez Gene ID: 3205083); a tap polypeptide from Mtb (Entrez Gene ID: 924773); a RV1473 polypeptide from Mtb (Entrez Gene ID: 886549); a katG polypeptide from Mtb (Entrez Gene ID: 885638); an inhA polypeptide from Mtb (Entrez Gene ID: 886523); a rpoB polypeptide from Mtb (Entrez Gene ID: 925979); a gidB polypeptide from Mtb (Entrez Gene ID: 886243); a pncA polypeptide from Mtb (Entrez Gene ID: 888260); an embB polypeptide from Mtb (Entrez Gene ID: 886126); and a gyrA polypeptide from Mtb (Entrez Gene ID: 887105). These polypeptides can include the wild-type sequences of these proteins or variants acquired with mutations that confer drug-resistance. In some cases, a vaccine provided herein can be designed to induce an immune response against a polypeptide involved in antibiotic resistance and one or more polypeptides not involved in antibiotic resistance (e.g., Ag85a of Mtb).
[0015] The vaccines provided herein can be used as stand alone vaccines or as adjuvants to other vaccines that target one or more pathogens (e.g., a vaccine that targets multiple Mtb antigens). In some cases, a vaccine provided herein (e.g., a vaccine that targets one or more polypeptides involved in antibiotic resistance) can be delivered in combination with another vaccine (e.g., a BCG vaccine). In some cases, nucleic acid encoding one or more polypeptides involved in antibiotic resistance can be inactivated and introduced into another vaccine (e.g., a BCG vaccine). Genetic engineering can be used to inactivate polypeptides involved in antibiotic resistance by mutating active site amino acids or by expressing the polypeptide as a cocktail of fragments of the full length polypeptide.
[0016] Any appropriate method can be used to deliver polypeptides involved in antibiotic resistance to a mammal so that an immune response is induced. For example, nucleic acid encoding a polypeptide involved in antibiotic resistance can be delivered using plasmids or viral vectors such as adenoviral vectors, vaccinia viral vectors, measles, or adeno-associated virus vectors. In some cases, the polypeptides can be produced in bacteria or yeast as purified polypeptide vaccines.
[0017] In some cases, a vaccine provided herein can be delivered as a prophylactic vaccine to assist in preventing the production of antibiotic resistance should an infection occur. In some cases, a vaccine provided herein can be applied therapeutically as an adjuvant to antibiotic treatment. For example, upon initiation of multidrug therapy against MDR or XDR, a patient can be vaccinated against Mtb and against one or more polypeptides involved in antibiotic resistance.
[0018] The vaccines provided herein can be designed to induce immune responses against one or more (e.g., two, three, four, five, six, seven, or more) polypeptides involved in antibiotic resistance. In some cases, a vaccine can be designed to be patient-specific. For example, a human known to have a particular antibiotic resistant pathogen can be given a vaccine that induces an immune response against the particular polypeptide or polypeptides involved in antibiotic resistance for that pathogen. Any appropriate method can be used to determine the identity of the particular polypeptides involved in antibiotic resistance for a particular pathogen including, without limitation, PCR techniques.
[0019] In some cases, polypeptides involved in antibiotic resistance can be used to generate antibodies (e.g., monoclonal antibodies) that can be used alone or in combination with antibiotics to deplete resistance proteins and enhance antibiotic efficacy.
[0020] In some cases, a vaccine provided herein (e.g., a vaccine that includes a recombinant polypeptide involve in antibiotic resistance) can be formulated with an adjuvant to form a composition for inducing an immune response when administered to a mammal. An adjuvant can be an immunological compound that can enhance an immune response against a particular antigen such as a polypeptide. Suitable adjuvants include, without limitation, alum as well as other aluminum-based compounds (e.g., Al2O3) that can be obtained from various commercial suppliers. For example, REHYDRAGEL® adjuvants can be obtained from Reheis Inc. (Berkeley Heights, N.J.). REHYDRAGEL® adjuvants are based on crystalline aluminum oxyhydroxide, and are hydrated gels containing crystalline particles with a large surface area (about 525 m2/g). Their Al2O3 content typically ranges from about 2 percent to about 10 percent. Rehydragel LG, for example, has an Al2O3 content of about 6 percent, and flows readily upon slight agitation. Rehydragel LG also has a protein binding capacity of 1.58 (i.e., 1.58 mg of bovine serum albumin bound per 1 mg of Al2O3), a sodium content of 0.02 percent, a chloride content of 0.28 percent, undetectable sulphate, an arsenic level less than 3 ppm, a heavy metal content less than 15 ppm, a pH of 6.5, and a viscosity of 1090 cp. Rehydragel LG can be combined with a polypeptide solution (e.g., a polypeptide in PBS) to yield Al(OH)3. In addition, ALHYDROGEL®, an aluminum hydroxy gel adjuvant, (Alhydrogel 1.3%, Alhydrogel 2.0%, or Alhydrogel "85") obtained from Brenntag Stinnes Logistics can be used.
[0021] In some cases, MN51 can be combined with a vaccine provided herein to form a composition that elicits an immune response when administered to a mammal. MN51 (MONTANIDE® Incomplete SEPPIC Adjuvant (USA) 51) as well as MN720 are available from Seppic (Paris, France). MN51 contains mannide oleate (MONTANIDE® 80, also known as anhydro mannitol octadecenoate) in mineral oil solution (Drakeol 6 VR). MONTANIDE® 80 is a limpid liquid with a maximum acid value of 1, a saponification value of 164-172, a hydroxyl value of 89-100, an iodine value of 67-75, a maximum peroxide value of 2, a heavy metal value less than 20 ppm, a maximum water content of 0.35%, a maximum color value of 9, and a viscosity at 25° C. of about 300 mPas. MONTANIDE® associated with oil (e.g., mineral oil, vegetable oil, squalane, squalene, or esters) is known as MONTANIDE® ISA. Drakeol 6 VR is a pharmaceutical grade mineral oil. Drakeol 6 VR contains no unsaturated or aromatic hydrocarbons, and has an A.P.I. gravity of 36.2-36.8, a specific gravity at 25° C. of 0.834-0.838, a viscosity at 100° F. of 59-61 SSU or 10.0-10.6 centistokes, a refractive index at 25° C. of 1.458-1.463, a better than minimum acid test, is negative for fluorescence at 360 nm, is negative for visible suspended matter, has an ASTM pour test value of 0-15° F., has a minimum ASTM flash point of 295° F., and complies with all RN requirements for light mineral oil and ultraviolet absorption. MN51 contains about 8 to 12 percent anhydro mannitol octadecenoate and about 88 to 92 percent mineral oil.
[0022] Other adjuvants include immuno-stimulating complexes (ISCOMs) that can contain such components as cholesterol and saponins ISCOM matrices can be prepared and conjugated to Cu2+ using methods such as those described herein. Adjuvants such as FCA, FIA, MN51, MN720, and Al(OH)3 are commercially available from companies such as Seppic, Difco Laboratories (Detroit, Mich.), and Superfos Biosector A/S (Vedbeak, Demark).
[0023] Other immunostimulatory components include, without limitation, muramyldipeptide (e.g., N-acetylmuramyl-L-alanyl-D-isoglutamine; MDP), monophosphoryl-lipid A (MPL), formyl-methionine containing tripeptides such as N-formyl-Met-Leu-Phe, or a bacterial lipopolysaccarhide. Such compounds are commercially available from Sigma Chemical Co. (St. Louis, Mo.) and RIBI ImmunoChem Research, Inc. (Hamilton, Mont.), for example. Additional immunostimulatory components can include pneumovax (an approved human vaccine), CD40L, or IL-12. In some embodiments, an adjuvant can be Complete Freund's Adjuvant or Incomplete Freund's Adjuvant.
[0024] This document also provides methods for preparing a vaccine provided herein. Such methods can involve suspending an amount of a nucleic acid vector (e.g., viral vector) or a polypeptide in a suitable amount of a physiological buffer (e.g., PBS). The nucleic acid vector or polypeptide then can be combined with a suitable amount of an adjuvant/immunostimulatory compound. The combining step can be achieved by any appropriate method, including, for example, stirring, shaking, vortexing, or passing back and forth through a needle attached to a syringe.
[0025] It is noted that the compositions can be prepared in batch, such that enough unit doses are obtained for multiple injections (e.g., injections into multiple mammals or multiple injections into the same mammal). A "unit dose" of a composition provided herein refers to the amount of a composition administered to a mammal at one time. A unit dose of the compositions provided herein can contain any amount of polypeptides involved in antibiotic resistance or nucleic acid encoding such polypeptides. For example, a unit dose of a composition can contain between about 0.1 μg and about 1 g (e.g., 1 μg, 10 μg, 15 μg, 25 μg, 30 μg, 50 μg, 100 μg, 250 μg, 280 μg, 300 μg, 500 μg, 750 μg, 1 mg, 10 mg, 15 mg, 25 mg, 30 mg, 50 mg, 100 mg, 250 mg, 280 mg, 300 mg, 500 mg, 750 mg, or more) of one or more polypeptides involved in antibiotic resistance. In the case of viral vectors, a unit dose of a composition can have a titer between about 103 to 1010 (e.g., 103, 104, 105, 106, 107, 108, 109, or 1010) viral particles or plaque forming units.
[0026] Methods for inducing a particular immune response in a mammal (e.g., a mouse, a rat, a cat, a dog, a horse, a cow, a non-human primate such as a cynomolgus monkey, or a human) include, without limitation, administering to a mammal an amount of a vaccine provided herein that is effective for producing an antibody response against one or more polypeptides involved in antibiotic resistance.
[0027] The vaccines provided herein can be administered using any appropriate method. Administration can be, for example, topical (e.g., transdermal, ophthalmic, or intranasal); pulmonary (e.g., by inhalation or insufflation of powders or aerosols); oral; or parenteral (e.g., by subcutaneous, intrathecal, intraventricular, intramuscular, or intraperitoneal injection, or by intravenous drip). Administration can be rapid (e.g., by injection) or can occur over a period of time (e.g., by slow infusion or administration of slow release formulations).
[0028] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
Example 1
Plasmids Containing Codon-optimized Antigens
[0029] Codon-optimized sequences for Mtb antigens are obtained from Genscript Corporation (Piscataway, N.J.) or generated using molecular cloning techniques. These sequences are codon-optimized for expression in mammalian cells for use as gene-based vaccines. The following Mtb genes that are expressed by H37Rv, MDR Mtb, or XDR Mtb or those listed in Table 1 that are expressed by H37Rv, MDR Mtb, or XDR Mtb are synthesized and cloned into the pShuttle-CMV plasmid:
[0030] Ag85a: Positive control protective antigen.
[0031] whiB7: Inducer of expression of a regulon of Mtb genes involved in antibiotic resistance (including tap, RV1473, and erm (Morris et al., Proc. Nat'l. Acad. Sci. USA, 102(34):12200-12205 (2005)).
[0032] tap: Drug efflux pump conferring low level resistance to aminoglyosides and tetracycline.
[0033] RV1473: Putative macrolide transporter induced by whiB7 (Morris et al., Proc. Nat'l. Acad. Sci. USA, 102(34):12200-12205 (2005)).
erm: Confers resistance to macrolide, lincosamide, and streptogramin.
TABLE-US-00001 TABLE 1 Genes involved in antibiotic resistance in Mycobacterium tuberculosis. Drug Resistance MDR XDR Inh katG (S315T) katG (S315T) Inh inhA -8TA inhA -8TA Rif rpoB (N487S) rpoB (D435Y, L452P) Streptomycin gidB Δ gidB Δ Pyrazinamide pncA (A132G) pncA Δ Ethambutol embB (M306V) embB (M306V) Ofloxacin gyrA A90V Kanamycin rrsA1401G
Where more than one mutant is shown in ( ), the other mutations are introduced by site-directed mutagenesis. Point mutations are likely irrelevant to generating cellular immunity since it is unlikely that these mutations will fall within MHC I or II epitopes.
[0034] Codon-optimized sequences for antigens from Staphylococcus aureus or other gram-positive bacteria, included those listed in Table 2, are generated using molecular cloning techniques. These sequences are codon-optimized for expression in mammalian cells for use as gene-based vaccines.
TABLE-US-00002 TABLE 2 Genes involved in antibiotic resistance in Staphylococcus aureus and other gram-positive bacteria. Drug Resistance MSSA MRSA VRSA Penicillin BlaZ BlaZ Methicillin -- MecA MecA Vancomycin -- VanS Vancomycin -- VanT MSSA is sensitive, penicillin-resistant S. aureus MRSA is methicillin-resistant S. aureus VRSA is vancomycin-resistant S. aureus
Example 2
Adenovirus Vectors Containing Codon-Optimized Antigens
[0035] Ad5 vectors are used to generate gene-based vaccines, which are used as an effective vaccine delivery vehicle in mice. Any appropriate vaccine carrier including Bacillus Calmette-Guerin (BCG) or vaccinia is used as a vaccine delivery vehicle in humans. In some cases, the recombinant polypeptides are delivered directly to the mammal (e.g., a human).
[0036] Once the pShuttle-CMV vectors are obtained, they are recombined into the Ad5 genome in bacteria and are used to generate CsCl-purified Ad5 vaccines.
Example 3
Codon-Optimized Gene for S. aureus BlaZ
[0037] The following codon-optimized nucleic acid sequence was generated to encode an S. aureus BlaZ polypeptide: AAGGAGCTGAACGACCTGGAGAAGAAGTACAACGCCC-ACATCGGCGTGTACGCCCTGGACACCAAGAGCGGC- AAGGAGGTGAAGTTCAACA GCGACAAGCGCTTCGCCTACGCCAGCACCAGCAAGGCCATCAACAGCGCCATCC TGCTGGAGCAGGTGCCCTACAACAAGCTGAACAAGAAGGTGCACATCAACAAGG ACGACATCGTGGCCTACAGCCCCATCCTGGAGAAGTACGTGGGCAAGGACATCA CCCTGAAGGCCCTGATCGAGGCCAGCATGACCTACAGCGACAACACCGCCAACA ACAAGATCATCAAGGAGATCGGCGGCATCAAGAAGGTGAAGCAGCGCCTGAAG GAGCTGGGCGACAAGGTGACCAACCCCGTGCGCTACGAGATCGAGCTGAACTAC TACAGCCCCAAGAGCAAGAAGGACACCAGCACCCCCGCCGCCTTCGGCAAGACC CTGAACAAGCTGATCGCCAACGGCAAGCTGAGCAAGGAGAACAAGAAGTTCCTG CTGGACCTGATGCTGAACAACAAGAGCGGCGACACCCTGATCAAGGACGGCGTG CCCAAGGACTACAAGGTGGCCGACAAGAGCGGCCAGGCCATCACCTACGCCAGC CGCAACGACGTGGCCTTCGTGTACCCCAAGGGCCAGAGCGAGCCCATCGTGCTG GTGATCTTCACCAACAAGGACAACAAGAGCGACAAGCCCAACGACAAGCTGATC AGCGAGACCGCCAAGAGCGTGATGAAGGAGTTC (SEQ ID NO:1). The amino acid sequence encoded by SEQ ID NO:1 is as follows:
TABLE-US-00003 (SEQ ID NO: 2) KELNDLEKKYNAHIGVYALDTKSGKEVKFNSDKRFAYASTSKAINSAIL LEQ-VPYNKLNKKVHINKDDIVAYSPILEKYVGKDITLKALIEASMTYS DNTANNKIIKEIGGIKKVKQRLKELGDKVTNPVRYEIELNYYSPKSKKD TSTPAAFGKTLNKLIANGKLSKENKKFLLDLMLNNKSGDTLIKDGVPKD YKVADKSGQAITYASRNDVAFVYPKGQSEPIVLVIFTNKDNKSDKPNDK LISETAKSVMKEF.
[0038] The codon-optimized nucleic acid encoding a BlaZ polypeptide was cloned into a shuttle plasmid fusing the sequence to the alpha 1-anti-trypsin secretory leader. Expression in cell lines generated detectable amounts of polypeptide from mammalian cells (FIG. 1).
[0039] Polypeptide vaccines can be produced from bacterial expression plasmids. The sequence of SEQ ID NO:1 was cloned into a pET-38 plasmid fusing it to a cellulose binding domain (CBD) and a His6 tag for purification. Expression of the polypeptide from bacteria followed by Western blotting revealed the presence of the polypeptide intracellularly in cell lysates and in a form secreted into the media (FIG. 2).
Example 4
Codon-Optimized Gene for S. aureus MecA
[0040] The following codon-optimized nucleic acid sequence was generated to encode an S. aureus MecA polypeptide: AGCAAGGACAAGGAGATCAACAACACCATCGACGC-CATCGAGGACAAGAACTTCAAGCAGGTGTACAAGGAC- AGCAGCTACATCAGCAA GAGCGACAACGGCGAGGTGGAGATGACCGAGCGCCCCATCAAGATCTACAACA GCCTGGGCGTGAAGGACATCAACATCCAGGACCGCAAGATCAAGAAGGTGAGCA AGAACAAGAAGCGCGTGGACGCCCAGTACAAGATCAAGACCAACTACGGCAAC ATCGACCGCAACGTGCAGTTCAACTTCGTGAAGGAGGACGGCATGTGGAAGCTG GACTGGGACCACAGCGTGATCATCCCCGGCATGCAGAAGGACCAGAGCATCCAC ATCGAGAACCTGAAGAGCGAGCGCGGCAAGATCCTGGACCGCAACAACGTGGA GCTGGCCAACACCGGCACCGCCTACGAGATCGGCATCGTGCCCAAGAACGTGAG CAAGAAGGACTACAAGGCCATCGCCAAGGAGCTGAGCATCAGCGAGGACTACAT CAAGCAGCAGATGGACCAGAACTGGGTGCAGGACGACACCTTCGTGCCCCTGAA GACCGTGAAGAAGATGGACGAGTACCTGAGCGACTTCGCCAAGAAGTTCCACCT GACCACCAACGAGACCGAGAGCCGCAACTACCCCCTGGGCAAGGCCACCAGCCA CCTGCTGGGCTACGTGGGCCCCATCAACAGCGAGGAGCTGAAGCAGAAGGAGTA CAAGGGCTACAAGGACGACGCCGTGATCGGCAAGAAGGGCCTGGAGAAGCTGT ACGACAAGAAGCTGCAGCACGAGGACGGCTACCGCGTGACCATCGTGGACGACA ACAGCAACACCATCGCCCACACCCTGATCGAGAAGAAGAAGAAGGACGGCAAG GACATCCAGCTGACCATCGACGCCAAGGTGCAGAAGAGCATCTACAACAACATG AAGAACGACTACGGCAGCGGCACCGCCATCCACCCCCAGACCGGCGAGCTGCTG GCCCTGGTGAGCACCCCCAGCTACGACGTGTACCCCTTCATGTACGGCATGAGCA ACGAGGAGTACAACAAGCTGACCGAGGACAAGAAGGAGCCCCTGCTGAACAAG TTCCAGATCACCACCAGCCCCGGCAGCACCCAGAAGATCCTGACCGCCATGATC GGCCTGAACAACAAGACCCTGGACGACAAGACCAGCTACAAGATCGACGGCAA GGGCTGGCAGAAGGACAAGAGCTGGGGCGGCTACAACGTGACCCGCTACGAGGT GGTGAACGGCAACATCGACCTGAAGCAGGCCATCGAGAGCAGCGACAACATCTT CTTCGCCCGCGTGGCCCTGGAGCTGGGCAGCAAGAAGTTCGAGAAGGGCATGAA GAAGCTGGGCGTGGGCGAGGACATCCCCAGCGACTACCCCTTCTACAACGCCCA GATCAGCAACAAGAACCTGGACAACGAGATCCTGCTGGCCGACAGCGGCTACGG CCAGGGCGAGATCCTGATCAACCCCGTGCAGATCCTGAGCATCTACAGCGCCCT GGAGAACAACGGCAACATCAACGCCCCCCACCTGCTGAAGGACACCAAGAACA AGGTGTGGAAGAAGAACATCATCAGCAAGGAGAACATCAACCTGCTGACCGACG GCATGCAGCAGGTGGTGAACAAGACCCACAAGGAGGACATCTACCGCAGCTACG CCAACCTGATCGGCAAGAGCGGCACCGCCGAGCTGAAGATGAAGCAGGGCGAG ACCGGCCGCCAGATCGGCTGGTTCATCAGCTACGACAAGGACAACCCCAACATG ATGATGGCCATCAACGTGAAGGACGTGCAGGACAAGGGCATGGCCAGCTACAAC GCCAAGATCAGCGGCAAGGTGTACGACGAGCTGTACGAGAACGGCAACAAGAA GTACGACATCGACGAG (SEQ ID NO:3). The amino acid sequence encoded by SEQ ID NO:3 is as follows: SKDK-EINNTIDAIEDKNFKQVYKDSSYISKSDNGEVEMTERPIKIYNSLGVKDINIQDRKIKK VSKNKKRVDAQYKIKTNYGNIDRNVQFNFVKEDGMWKLDWDHSVIIPGMQKDQSI HIENLKSERGKILDRNNVELANTGTAYEIGIVPKNVSKKDYKAIAKELSISEDYIKQQ MDQNWVQDDTFVPLKTVKKMDEYLSDFAKKFHLTTNETESRNYPLGKATSHLLGY VGPINSEELKQKEYKGYKDDAVIGKKGLEKLYDKKLQHEDGYRVTIVDDNSNTIAH TLIEKKKKDGKDIQLTIDAKVQKSIYNNMKNDYGSGTAIHPQTGELLALVSTPSYDV YPFMYGMSNEEYNKLTEDKKEPLLNKFQITTSPGSTQKILTAMIGLNNKTLDDKTSY KIDGKGWQKDKSWGGYNVTRYEVVNGNIDLKQAIESSDNIFFARVALELGSKKFEK GMKKLGVGEDIPSDYPFYNAQISNKNLDNEILLADSGYGQGEILINPVQILSIYSALEN NGNINAPHLLKDTKNKVWKKNIISKENINLLTDGMQQVVNKTHKEDIYRSYANLIGK SGTAELKMKQGETGRQIGWFISYDKDNPNMMMAINVKDVQDKGMASYNAKISGKV YDELYENGNKKYDIDE (SEQ ID NO: 4).
[0041] The codon-optimized nucleic acid encoding a MecA polypeptide was cloned into a shuttle plasmid fusing the sequence to the alpha 1-anti-trypsin secretory leader. Expression in cell lines generated detectable amounts of polypeptide from mammalian cells (FIG. 1).
Example 5
Administration of Vaccine
[0042] Separate groups of 50 BALB/c mice are immunized intranasally with 1010 virus particles of Ad vaccines expressing the following antigens: saline; GFP (negative control vaccine for Mtb); Ag85a (positive control for CD8 responses and protection); and whiB7, tap, RV1473 and erm (vaccines against polypeptides involved in antibiotic resistance). Additional groups are used for the genes listed in Table 1. Four weeks later, five mice from each group are evaluated for cellular immune responses using, e.g., ELISPOT, intracellular cytokine staining. At the same time, the remaining mice of each group are challenged with 300 colony forming units of H37Rv/mouse by aerosolization to mimic the normal Mtb infection route. H37Rv is not classified as MDR or XDR, but is resistant to a number of antibiotics (Morris et al., Proc. Nat'l. Acad. Sci. USA, 102(34):12200-12205 (2005)). In a separate study, legitimate MDR or XDR Mtb is used in place of H37Rv. At 2, 4, 16, and 32 weeks post-infection (WPI), ten mice from each group are sacrificed, and Mtb is titered from the lung and the spleen. Histopathology and MST testing are also performed on the samples. The remaining mice are retained to estimate median survival time (MST). Control animals typically survive until 30 weeks with longer survival for successful vaccines. At 48 weeks, all animals are sacrificed for titering and histopathology.
Other Embodiments
[0043] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
Sequence CWU
1
41771DNAStaphylococcus aureus 1aaggagctga acgacctgga gaagaagtac aacgcccaca
tcggcgtgta cgccctggac 60accaagagcg gcaaggaggt gaagttcaac agcgacaagc
gcttcgccta cgccagcacc 120agcaaggcca tcaacagcgc catcctgctg gagcaggtgc
cctacaacaa gctgaacaag 180aaggtgcaca tcaacaagga cgacatcgtg gcctacagcc
ccatcctgga gaagtacgtg 240ggcaaggaca tcaccctgaa ggccctgatc gaggccagca
tgacctacag cgacaacacc 300gccaacaaca agatcatcaa ggagatcggc ggcatcaaga
aggtgaagca gcgcctgaag 360gagctgggcg acaaggtgac caaccccgtg cgctacgaga
tcgagctgaa ctactacagc 420cccaagagca agaaggacac cagcaccccc gccgccttcg
gcaagaccct gaacaagctg 480atcgccaacg gcaagctgag caaggagaac aagaagttcc
tgctggacct gatgctgaac 540aacaagagcg gcgacaccct gatcaaggac ggcgtgccca
aggactacaa ggtggccgac 600aagagcggcc aggccatcac ctacgccagc cgcaacgacg
tggccttcgt gtaccccaag 660ggccagagcg agcccatcgt gctggtgatc ttcaccaaca
aggacaacaa gagcgacaag 720cccaacgaca agctgatcag cgagaccgcc aagagcgtga
tgaaggagtt c 7712257PRTStaphylococcus aureus 2Lys Glu Leu Asn
Asp Leu Glu Lys Lys Tyr Asn Ala His Ile Gly Val1 5
10 15Tyr Ala Leu Asp Thr Lys Ser Gly Lys Glu
Val Lys Phe Asn Ser Asp 20 25
30Lys Arg Phe Ala Tyr Ala Ser Thr Ser Lys Ala Ile Asn Ser Ala Ile
35 40 45Leu Leu Glu Gln Val Pro Tyr Asn
Lys Leu Asn Lys Lys Val His Ile 50 55
60Asn Lys Asp Asp Ile Val Ala Tyr Ser Pro Ile Leu Glu Lys Tyr Val65
70 75 80Gly Lys Asp Ile Thr
Leu Lys Ala Leu Ile Glu Ala Ser Met Thr Tyr 85
90 95Ser Asp Asn Thr Ala Asn Asn Lys Ile Ile Lys
Glu Ile Gly Gly Ile 100 105
110Lys Lys Val Lys Gln Arg Leu Lys Glu Leu Gly Asp Lys Val Thr Asn
115 120 125Pro Val Arg Tyr Glu Ile Glu
Leu Asn Tyr Tyr Ser Pro Lys Ser Lys 130 135
140Lys Asp Thr Ser Thr Pro Ala Ala Phe Gly Lys Thr Leu Asn Lys
Leu145 150 155 160Ile Ala
Asn Gly Lys Leu Ser Lys Glu Asn Lys Lys Phe Leu Leu Asp
165 170 175Leu Met Leu Asn Asn Lys Ser
Gly Asp Thr Leu Ile Lys Asp Gly Val 180 185
190Pro Lys Asp Tyr Lys Val Ala Asp Lys Ser Gly Gln Ala Ile
Thr Tyr 195 200 205Ala Ser Arg Asn
Asp Val Ala Phe Val Tyr Pro Lys Gly Gln Ser Glu 210
215 220Pro Ile Val Leu Val Ile Phe Thr Asn Lys Asp Asn
Lys Ser Asp Lys225 230 235
240Pro Asn Asp Lys Leu Ile Ser Glu Thr Ala Lys Ser Val Met Lys Glu
245 250 255Phe
31932DNAStaphylococcus aureus 3agcaaggaca aggagatcaa caacaccatc
gacgccatcg aggacaagaa cttcaagcag 60gtgtacaagg acagcagcta catcagcaag
agcgacaacg gcgaggtgga gatgaccgag 120cgccccatca agatctacaa cagcctgggc
gtgaaggaca tcaacatcca ggaccgcaag 180atcaagaagg tgagcaagaa caagaagcgc
gtggacgccc agtacaagat caagaccaac 240tacggcaaca tcgaccgcaa cgtgcagttc
aacttcgtga aggaggacgg catgtggaag 300ctggactggg accacagcgt gatcatcccc
ggcatgcaga aggaccagag catccacatc 360gagaacctga agagcgagcg cggcaagatc
ctggaccgca acaacgtgga gctggccaac 420accggcaccg cctacgagat cggcatcgtg
cccaagaacg tgagcaagaa ggactacaag 480gccatcgcca aggagctgag catcagcgag
gactacatca agcagcagat ggaccagaac 540tgggtgcagg acgacacctt cgtgcccctg
aagaccgtga agaagatgga cgagtacctg 600agcgacttcg ccaagaagtt ccacctgacc
accaacgaga ccgagagccg caactacccc 660ctgggcaagg ccaccagcca cctgctgggc
tacgtgggcc ccatcaacag cgaggagctg 720aagcagaagg agtacaaggg ctacaaggac
gacgccgtga tcggcaagaa gggcctggag 780aagctgtacg acaagaagct gcagcacgag
gacggctacc gcgtgaccat cgtggacgac 840aacagcaaca ccatcgccca caccctgatc
gagaagaaga agaaggacgg caaggacatc 900cagctgacca tcgacgccaa ggtgcagaag
agcatctaca acaacatgaa gaacgactac 960ggcagcggca ccgccatcca cccccagacc
ggcgagctgc tggccctggt gagcaccccc 1020agctacgacg tgtacccctt catgtacggc
atgagcaacg aggagtacaa caagctgacc 1080gaggacaaga aggagcccct gctgaacaag
ttccagatca ccaccagccc cggcagcacc 1140cagaagatcc tgaccgccat gatcggcctg
aacaacaaga ccctggacga caagaccagc 1200tacaagatcg acggcaaggg ctggcagaag
gacaagagct ggggcggcta caacgtgacc 1260cgctacgagg tggtgaacgg caacatcgac
ctgaagcagg ccatcgagag cagcgacaac 1320atcttcttcg cccgcgtggc cctggagctg
ggcagcaaga agttcgagaa gggcatgaag 1380aagctgggcg tgggcgagga catccccagc
gactacccct tctacaacgc ccagatcagc 1440aacaagaacc tggacaacga gatcctgctg
gccgacagcg gctacggcca gggcgagatc 1500ctgatcaacc ccgtgcagat cctgagcatc
tacagcgccc tggagaacaa cggcaacatc 1560aacgcccccc acctgctgaa ggacaccaag
aacaaggtgt ggaagaagaa catcatcagc 1620aaggagaaca tcaacctgct gaccgacggc
atgcagcagg tggtgaacaa gacccacaag 1680gaggacatct accgcagcta cgccaacctg
atcggcaaga gcggcaccgc cgagctgaag 1740atgaagcagg gcgagaccgg ccgccagatc
ggctggttca tcagctacga caaggacaac 1800cccaacatga tgatggccat caacgtgaag
gacgtgcagg acaagggcat ggccagctac 1860aacgccaaga tcagcggcaa ggtgtacgac
gagctgtacg agaacggcaa caagaagtac 1920gacatcgacg ag
19324644PRTStaphylococcus aureus 4Ser
Lys Asp Lys Glu Ile Asn Asn Thr Ile Asp Ala Ile Glu Asp Lys1
5 10 15Asn Phe Lys Gln Val Tyr Lys
Asp Ser Ser Tyr Ile Ser Lys Ser Asp 20 25
30Asn Gly Glu Val Glu Met Thr Glu Arg Pro Ile Lys Ile Tyr
Asn Ser 35 40 45Leu Gly Val Lys
Asp Ile Asn Ile Gln Asp Arg Lys Ile Lys Lys Val 50 55
60Ser Lys Asn Lys Lys Arg Val Asp Ala Gln Tyr Lys Ile
Lys Thr Asn65 70 75
80Tyr Gly Asn Ile Asp Arg Asn Val Gln Phe Asn Phe Val Lys Glu Asp
85 90 95Gly Met Trp Lys Leu Asp
Trp Asp His Ser Val Ile Ile Pro Gly Met 100
105 110Gln Lys Asp Gln Ser Ile His Ile Glu Asn Leu Lys
Ser Glu Arg Gly 115 120 125Lys Ile
Leu Asp Arg Asn Asn Val Glu Leu Ala Asn Thr Gly Thr Ala 130
135 140Tyr Glu Ile Gly Ile Val Pro Lys Asn Val Ser
Lys Lys Asp Tyr Lys145 150 155
160Ala Ile Ala Lys Glu Leu Ser Ile Ser Glu Asp Tyr Ile Lys Gln Gln
165 170 175Met Asp Gln Asn
Trp Val Gln Asp Asp Thr Phe Val Pro Leu Lys Thr 180
185 190Val Lys Lys Met Asp Glu Tyr Leu Ser Asp Phe
Ala Lys Lys Phe His 195 200 205Leu
Thr Thr Asn Glu Thr Glu Ser Arg Asn Tyr Pro Leu Gly Lys Ala 210
215 220Thr Ser His Leu Leu Gly Tyr Val Gly Pro
Ile Asn Ser Glu Glu Leu225 230 235
240Lys Gln Lys Glu Tyr Lys Gly Tyr Lys Asp Asp Ala Val Ile Gly
Lys 245 250 255Lys Gly Leu
Glu Lys Leu Tyr Asp Lys Lys Leu Gln His Glu Asp Gly 260
265 270Tyr Arg Val Thr Ile Val Asp Asp Asn Ser
Asn Thr Ile Ala His Thr 275 280
285Leu Ile Glu Lys Lys Lys Lys Asp Gly Lys Asp Ile Gln Leu Thr Ile 290
295 300Asp Ala Lys Val Gln Lys Ser Ile
Tyr Asn Asn Met Lys Asn Asp Tyr305 310
315 320Gly Ser Gly Thr Ala Ile His Pro Gln Thr Gly Glu
Leu Leu Ala Leu 325 330
335Val Ser Thr Pro Ser Tyr Asp Val Tyr Pro Phe Met Tyr Gly Met Ser
340 345 350Asn Glu Glu Tyr Asn Lys
Leu Thr Glu Asp Lys Lys Glu Pro Leu Leu 355 360
365Asn Lys Phe Gln Ile Thr Thr Ser Pro Gly Ser Thr Gln Lys
Ile Leu 370 375 380Thr Ala Met Ile Gly
Leu Asn Asn Lys Thr Leu Asp Asp Lys Thr Ser385 390
395 400Tyr Lys Ile Asp Gly Lys Gly Trp Gln Lys
Asp Lys Ser Trp Gly Gly 405 410
415Tyr Asn Val Thr Arg Tyr Glu Val Val Asn Gly Asn Ile Asp Leu Lys
420 425 430Gln Ala Ile Glu Ser
Ser Asp Asn Ile Phe Phe Ala Arg Val Ala Leu 435
440 445Glu Leu Gly Ser Lys Lys Phe Glu Lys Gly Met Lys
Lys Leu Gly Val 450 455 460Gly Glu Asp
Ile Pro Ser Asp Tyr Pro Phe Tyr Asn Ala Gln Ile Ser465
470 475 480Asn Lys Asn Leu Asp Asn Glu
Ile Leu Leu Ala Asp Ser Gly Tyr Gly 485
490 495Gln Gly Glu Ile Leu Ile Asn Pro Val Gln Ile Leu
Ser Ile Tyr Ser 500 505 510Ala
Leu Glu Asn Asn Gly Asn Ile Asn Ala Pro His Leu Leu Lys Asp 515
520 525Thr Lys Asn Lys Val Trp Lys Lys Asn
Ile Ile Ser Lys Glu Asn Ile 530 535
540Asn Leu Leu Thr Asp Gly Met Gln Gln Val Val Asn Lys Thr His Lys545
550 555 560Glu Asp Ile Tyr
Arg Ser Tyr Ala Asn Leu Ile Gly Lys Ser Gly Thr 565
570 575Ala Glu Leu Lys Met Lys Gln Gly Glu Thr
Gly Arg Gln Ile Gly Trp 580 585
590Phe Ile Ser Tyr Asp Lys Asp Asn Pro Asn Met Met Met Ala Ile Asn
595 600 605Val Lys Asp Val Gln Asp Lys
Gly Met Ala Ser Tyr Asn Ala Lys Ile 610 615
620Ser Gly Lys Val Tyr Asp Glu Leu Tyr Glu Asn Gly Asn Lys Lys
Tyr625 630 635 640Asp Ile
Asp Glu
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