METHODS AND MATERIALS FOR PRODUCING IMMUNE RESPONSES AGAINST POLYPEPTIDES INVOLVED IN ANTIBIOTIC RESISTANCE

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.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No. 13/001,228, filed Feb. 18, 2011, which is a National Stage Application under 35 U.S.C. §371 of International Application No. PCT/US2009/048018, filed Jun. 19, 2009, which claims benefit of priority from U.S. Provisional Application Ser. No. 61/075,881, filed on Jun. 26, 2008. The disclosures of the prior applications are considered part of (and are incorporated by reference in) the disclosure of this application.

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 (ISA) 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), CD4OL, 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)).

[0034] 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.

[0035] 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

[0036] 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).

[0037] Once the pShuttle-CMV vectors are obtained, they are recombined into the Ad5 genome in bacteria and are used to generate CsC1-purified Ad5 vaccines.

Example 3

Codon-optimized Gene for S. aureus BlaZ

[0038] The following codon-optimized nucleic acid sequence was generated to encode an S. aureus B1aZ 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: KELNDLEKKYNAHIGVYALDTKSGKEVKFNSDKRFAYASTSKAINSAILLEQ-VPYNKLNKKVHINKDDIVAY- SPILEKYVGKDITLKALIEASMTYSDNTANNKIIKEIG GIKKVKQRLKELGDKVTNPVRYEIELNYYSPKSKKDTSTPAAFGKTLNKLIANGKLS KENKKFLLDLMLNNKSGDTLIKDGVPKDYKVADKSGQAITYASRNDVAFVYPKGQS EPIVLVIFTNKDNKSDKPNDKLISETAKSVMKEF (SEQ ID NO:2).

[0039] 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).

[0040] Polypeptide vaccines can be produced from bacterial expression plasmids. The sequence of SEQ ID NO:1 was cloned into a pET-3 8 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

[0041] 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).

[0042] 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

[0043] 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 vaccinees. At 48 weeks, all animals are sacrificed for titering and histopathology.

Other Embodiments

[0044] 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

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 15 Tyr Ala Leu Asp Thr Lys Ser Gly Lys Glu Val Lys Phe Asn Ser Asp 20 25 30 Lys Arg Phe Ala Tyr Ala Ser Thr Ser Lys Ala Ile Asn Ser Ala Ile 35 40 45 Leu Leu Glu Gln Val Pro Tyr Asn Lys Leu Asn Lys Lys Val His Ile 50 55 60 Asn Lys Asp Asp Ile Val Ala Tyr Ser Pro Ile Leu Glu Lys Tyr Val65 70 75 80 Gly Lys Asp Ile Thr Leu Lys Ala Leu Ile Glu Ala Ser Met Thr Tyr 85 90 95 Ser Asp Asn Thr Ala Asn Asn Lys Ile Ile Lys Glu Ile Gly Gly Ile 100 105 110 Lys Lys Val Lys Gln Arg Leu Lys Glu Leu Gly Asp Lys Val Thr Asn 115 120 125 Pro Val Arg Tyr Glu Ile Glu Leu Asn Tyr Tyr Ser Pro Lys Ser Lys 130 135 140 Lys Asp Thr Ser Thr Pro Ala Ala Phe Gly Lys Thr Leu Asn Lys Leu145 150 155 160 Ile Ala Asn Gly Lys Leu Ser Lys Glu Asn Lys Lys Phe Leu Leu Asp 165 170 175 Leu Met Leu Asn Asn Lys Ser Gly Asp Thr Leu Ile Lys Asp Gly Val 180 185 190 Pro Lys Asp Tyr Lys Val Ala Asp Lys Ser Gly Gln Ala Ile Thr Tyr 195 200 205 Ala Ser Arg Asn Asp Val Ala Phe Val Tyr Pro Lys Gly Gln Ser Glu 210 215 220 Pro Ile Val Leu Val Ile Phe Thr Asn Lys Asp Asn Lys Ser Asp Lys225 230 235 240 Pro Asn Asp Lys Leu Ile Ser Glu Thr Ala Lys Ser Val Met Lys Glu 245 250 255 Phe31932DNAStaphylococcus 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 15 Asn Phe Lys Gln Val Tyr Lys Asp Ser Ser Tyr Ile Ser Lys Ser Asp 20 25 30 Asn Gly Glu Val Glu Met Thr Glu Arg Pro Ile Lys Ile Tyr Asn Ser 35 40 45 Leu Gly Val Lys Asp Ile Asn Ile Gln Asp Arg Lys Ile Lys Lys Val 50 55 60 Ser Lys Asn Lys Lys Arg Val Asp Ala Gln Tyr Lys Ile Lys Thr Asn65 70 75 80 Tyr Gly Asn Ile Asp Arg Asn Val Gln Phe Asn Phe Val Lys Glu Asp 85 90 95 Gly Met Trp Lys Leu Asp Trp Asp His Ser Val Ile Ile Pro Gly Met 100 105 110 Gln Lys Asp Gln Ser Ile His Ile Glu Asn Leu Lys Ser Glu Arg Gly 115 120 125 Lys Ile Leu Asp Arg Asn Asn Val Glu Leu Ala Asn Thr Gly Thr Ala 130 135 140 Tyr Glu Ile Gly Ile Val Pro Lys Asn Val Ser Lys Lys Asp Tyr Lys145 150 155 160 Ala Ile Ala Lys Glu Leu Ser Ile Ser Glu Asp Tyr Ile Lys Gln Gln 165 170 175 Met Asp Gln Asn Trp Val Gln Asp Asp Thr Phe Val Pro Leu Lys Thr 180 185 190 Val Lys Lys Met Asp Glu Tyr Leu Ser Asp Phe Ala Lys Lys Phe His 195 200 205 Leu Thr Thr Asn Glu Thr Glu Ser Arg Asn Tyr Pro Leu Gly Lys Ala 210 215 220 Thr Ser His Leu Leu Gly Tyr Val Gly Pro Ile Asn Ser Glu Glu Leu225 230 235 240 Lys Gln Lys Glu Tyr Lys Gly Tyr Lys Asp Asp Ala Val Ile Gly Lys 245 250 255 Lys Gly Leu Glu Lys Leu Tyr Asp Lys Lys Leu Gln His Glu Asp Gly 260 265 270 Tyr Arg Val Thr Ile Val Asp Asp Asn Ser Asn Thr Ile Ala His Thr 275 280 285 Leu Ile Glu Lys Lys Lys Lys Asp Gly Lys Asp Ile Gln Leu Thr Ile 290 295 300 Asp Ala Lys Val Gln Lys Ser Ile Tyr Asn Asn Met Lys Asn Asp Tyr305 310 315 320 Gly Ser Gly Thr Ala Ile His Pro Gln Thr Gly Glu Leu Leu Ala Leu 325 330 335 Val Ser Thr Pro Ser Tyr Asp Val Tyr Pro Phe Met Tyr Gly Met Ser 340 345 350 Asn Glu Glu Tyr Asn Lys Leu Thr Glu Asp Lys Lys Glu Pro Leu Leu 355 360 365 Asn Lys Phe Gln Ile Thr Thr Ser Pro Gly Ser Thr Gln Lys Ile Leu 370 375 380 Thr Ala Met Ile Gly Leu Asn Asn Lys Thr Leu Asp Asp Lys Thr Ser385 390 395 400 Tyr Lys Ile Asp Gly Lys Gly Trp Gln Lys Asp Lys Ser Trp Gly Gly 405 410 415 Tyr Asn Val Thr Arg Tyr Glu Val Val Asn Gly Asn Ile Asp Leu Lys 420 425 430 Gln Ala Ile Glu Ser Ser Asp Asn Ile Phe Phe Ala Arg Val Ala Leu 435 440 445 Glu Leu Gly Ser Lys Lys Phe Glu Lys Gly Met Lys Lys Leu Gly Val 450 455 460 Gly Glu Asp Ile Pro Ser Asp Tyr Pro Phe Tyr Asn Ala Gln Ile Ser465 470 475 480 Asn Lys Asn Leu Asp Asn Glu Ile Leu Leu Ala Asp Ser Gly Tyr Gly 485 490 495 Gln Gly Glu Ile Leu Ile Asn Pro Val Gln Ile Leu Ser Ile Tyr Ser 500 505 510 Ala Leu Glu Asn Asn Gly Asn Ile Asn Ala Pro His Leu Leu Lys Asp 515 520 525 Thr Lys Asn Lys Val Trp Lys Lys Asn Ile Ile Ser Lys Glu Asn Ile 530 535 540 Asn Leu Leu Thr Asp Gly Met Gln Gln Val Val Asn Lys Thr His Lys545 550 555 560 Glu Asp Ile Tyr Arg Ser Tyr Ala Asn Leu Ile Gly Lys Ser Gly Thr 565 570 575 Ala Glu Leu Lys Met Lys Gln Gly Glu Thr Gly Arg Gln Ile Gly Trp 580 585 590 Phe Ile Ser Tyr Asp Lys Asp Asn Pro Asn Met Met Met Ala Ile Asn 595 600 605 Val Lys Asp Val Gln Asp Lys Gly Met Ala Ser Tyr Asn Ala Lys Ile 610 615 620 Ser Gly Lys Val Tyr Asp Glu Leu Tyr Glu Asn Gly Asn Lys Lys Tyr625 630 635 640 Asp Ile Asp Glu

Claims

1-15. (canceled)

16. A method for inducing an immune response against an intracellular pathogen that is resistant to an antibiotic in a human having an infection with said intracellular pathogen, wherein said method comprises administering to said human an amount of a polypeptide or a nucleic acid encoding said polypeptide effective for producing said immune response in said human that reduces the severity of said infection within said human, wherein said polypeptide is involved in said antibiotic resistance, and wherein said intracellular pathogen is a Mycobacterium tuberculosis pathogen.

17. The method of claim 16, wherein said method comprises administering said polypeptide to said human.

18. The method of claim 16, wherein said method comprises administering said nucleic acid to said human.

19. The method of claim 18, wherein said nucleic acid is a viral vector encoding said polypeptide.

20. The method of claim 19, wherein said viral vector is an adenoviral, vaccinia viral, measles, or adeno-associated virus vector.

21. The method of claim 16, wherein said polypeptide is 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.

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