METHODS AND COMPOSITIONS TO PREVENT OR TREAT BACTERIAL INFECTIONS

Abstract

Certain embodiments are directed to methods and compositions for preventing treating bacterial infections. In certain embodiments the compositions comprise thioredoxin deficient bacteria.

Claims

1. A method of treating or preventing colonization, infection, or disease by a Acinetobacter baumannii microbe comprising administering a clinically effective dose of an attenuated Acinetobacter baumannii to a subject in need thereof.

2. The method of claim 1, wherein the attenuated Acinetobacter baumannii is deficient in thioredoxin-A.

3. The method of claim 1, wherein the attenuated Acinetobacter baumannii is administered before the administration of an antimicrobial agent.

4. The method of claim 1, wherein the attenuated Acinetobacter baumannii is administered orally.

5. The method of claim 1, wherein the attenuated Acinetobacter baumannii is administered as live attenuated Acinetobacter baumannii.

6. A Acinetobacter baumannii, wherein the Acinetobacter baumannii is deficient in thioredoxin-A.

7. A vaccine comprising attenuated Acinetobacter baumannii, wherein the attenuated Acinetobacter baumannii is Acinetobacter baumannii deficient in thioredoxin-A.

8. The vaccine of claim 7, wherein the attenuated Acinetobacter baumannii is live attenuated Acinetobacter baumannii.

9. The vaccine of claim 7, wherein the vaccine is formulated for oral administration.

10. The vaccine of claim 7, wherein the vaccine is formulated for vaccination against Acinetobacter baumannii.

Description

DESCRIPTION OF THE DRAWINGS

[0038] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.

[0039] FIG. 1 shows Acinetobacter baumannii dissociates Secretory Component (SC) from SIgA through a reductive process.

[0040] FIG. 2 shows IgA enhances virulence of Acinetobacter baumannii during GI challenge.

[0041] FIG. 3 shows IgA enhances Acinetobacter baumannii adherence and colonization in the GI tract.

[0042] FIG. 4 is a working model of Acinetobacter baumannii gastrointestinal infection.

[0043] FIG. 5 shows SIgA reduction and intestinal adhesion by Acinetobacter baumannii inhibited by thioredoxin inhibitor PX-12.

[0044] FIG. 6 shows the antimicrobial effect of PX-12 on Acinetobacter baumannii.

[0045] FIG. 7 shows treatment with the mammalian thioredoxin-1 inhibitor PX-12 significantly reduced bacterial attachment by 40%.

[0046] FIG. 8 shows thioredoxin as a mediator of SIgA breakdown

[0047] FIG. 9 shows reduction of SIgA by recombinant A. baumannii thioredoxin A (abTrxA).

[0048] FIG. 10 shows a repeat minimum inhibitory concentration (MIC) experiment performed to CLSI standards: Mueller Hinton broth cation adjusted (Ca.sup.++25 mg/L, Mg.sup.++12.5 mg/L), bacterial concentration reduced to 510.sup.5 CFU/mL, compound concentrations tested range from 64 to 0.5 g/mL, low MIC combined with low MBC indicates PX-12 compound has bactericidal activity.

[0049] FIG. 11 shows a method used for producing attenuated Acinetobacter baumannii deficient in TrxA (trxA).

[0050] FIG. 12 shows that the virulence of attenuated Acinetobacter baumannii is decreased in comparison to wild type (WT) strains in mice.

[0051] FIG. 13 shows that surviving mice from the sepsis challenge showed increased survival rates in a secondary sepsis challenge.

[0052] FIG. 14 shows an i.p. vaccination sepsis model used to test the effectiveness of live attenuated Acinetobacter baumannii as a vaccine.

[0053] FIG. 15 shows reduced virulence of attenuated Acinetobacter baumannii compared to WT.

[0054] FIG. 16 shows Ci79 specific and non-specific humoral responses to a primary Acinetobacter baumannii sepsis.

[0055] FIG. 17 shows increased survival rates by primary Acinetobacter baumannii sepsis challenge surviving mice in a secondary sepsis challenge.

[0056] FIG. 18 shows total Ig bound to Ci79 coated plates from blood taken from mice tested in a secondary Acinetobacter baumannii sepsis challenge.

[0057] FIG. 19 shows total Ig bound to TrxA coated plates from blood taken from the mice tested in a secondary Acinetobacter baumannii sepsis challenge.

[0058] FIG. 20 shows methodology that can be used to test for Ig against Ci79 over time after initial inoculation and to test survival of mice exposed to a secondary Acinetobacter baumannii sepsis challenge using a 100% LD.sub.50 amount of Acinetobacter baumannii.

[0059] FIG. 21A-21B shows survival/virulence results upon bacterial challenge. (A) wild typ Ci79 survival. (B) AtrxA survival.

[0060] FIG. 22 shows the results of a secondary challenge with 2 LD50 of wildtype bacteria.

[0061] FIG. 23 shows antibody response (A) 28 days post vaccination and (B) 14 days post-secondary challenge. (C) illustration of trend line.

[0062] FIG. 24 shows the results of a high challenge dose of 10 LD50 of wildtype Ci79.

[0063] FIG. 25 shows (A) organ burden and (B) PTX3 production after high challenge dose of 10 LD50 of wildtype Ci79.

[0064] FIG. 26 shows pathology scores for (A) liver and (B) spleen from high challenge dose of 10 LD50 of wildtype Ci79.

[0065] FIG. 27 shows antibody isotyping for (A) animals vaccinated with wildtype and (B) animals vaccinated with mutant at 14 and 28 days.

[0066] FIG. 28 shows the T cell mediated responses (A) IL-4, (B) IFN, and (C) IL-17.

[0067] FIG. 29 shows results in the form of (A) survival and (B) antibody response for vaccination via subcutaneous administration.

DESCRIPTION

[0068] Immunoglobulin A (IgA) is an antibody that plays a critical role in mucosal immunity. IgA can exist in a dimeric form, which is called secretory IgA (SIgA). In its secretory form, IgA is the main immunoglobulin found in mucosal secretions, including tears, saliva, colostrum and secretions from the genitourinary tract, gastrointestinal tract, prostate and respiratory epithelium. The secretory component of SIgA protects the immunoglobulin from being degraded by proteolytic enzymes, thus SIgA can survive in the harsh gastrointestinal tract environment and provide protection against microbes that multiply in body secretions. Case studies and recent literature suggest a potential link between gastrointestinal (GI) colonization and acquired antimicrobial resistance potentially following Acinetobacter baumannii breakdown of SIgA. Breakdown of SIgA has been shown to have an immunosuppressive effect, due to the liberation of secretory component (SC) from SIgA and subsequent inhibition of neutrophil recruitment (Mantis et al. Annals of Internal Medicine, 1998. 129(3):182-189). The breakdown of SIgA also aids bacteria in colonization of the intestinal epithelium. The inventors have conducted studies to investigate how Acinetobacter baumannii breaks down SIgA and what affect this has on the virulence of the organism in vivo. The data show that SIgA breakdown by Acinetobacter baumannii is a reductive process, rather than a proteolytic, and is significantly reduced after addition of the thioredoxin colorimetric substrate DTNB, suggesting it is acting as a competitive inhibitor.

[0069] Prevention of Acinetobacter baumannii infections is desirable. Inactivated whole cell vaccine as well as OmpA (Omp38) subunit vaccines have shown therapeutic potential. However, neither are approved for use in humans. Herein, the inventors have created a new attenuated Acinetobacter baumannii mutant from a multi-drug resistant clinical isolate deficient in thioredoxin-A (TrxA). This organism exhibits markedly reduced virulence in an i.p. sepsis model and has the potential for use as a live vaccine against Acinetobacter baumannii infection.

I. Acinetobacter

[0070] Acinetobacter is a genus of Gram-negative bacteria belonging to the Gammaproteobacteria. Acinetobacter spp. are non-motile and oxidase-negative, and occur in pairs under magnification. They are important soil organisms, where they contribute to the mineralization of, for example, aromatic compounds. Acinetobacter spp. are a source of infection in debilitated patients in the hospital, in particular the species Acinetobacter baumannii. Species of the genus Acinetobacter are aerobic non-fermentative Gram-negative bacilli. Most strains of Acinetobacter, except some of the A. lwoffii strain, grow well on MacConkey agar (without salt). Although officially classified as nonlactose-fermenting, they are often partially lactose-fermenting when grown on MacConkey agar. They are oxidase-negative, nonmotile, and usually nitrate negative. Bacteria of the genus Acinetobacter are known to form intracellular inclusions of polyhydroxyalkanoates under certain environmental conditions.

[0071] FIG. 1 shows Acinetobacter baumannii dissociates Secretory Component (SC) from SIgA through a reductive process. Acinetobacter baumannii was incubated with 50 g/mL SIgA to examine breakdown of the immunoglobulin. Breakdown of SIgA by A. baumannii was observed by liberation of secretory component from dimeric IgA. Goat anti-human secretory component (free and bound) antibody was used to monitor this occurrence though Western blot. A mixture of A. baumannii strain Ci79 was prepared in three concentrations 10.sup.7, 10.sup.6, and 10.sup.5 CFU/mLto determine whether SIgA breakdown was dose dependent with respect to bacterial concentration FIG. 1A. Previous literature examining SIgA degradation by Gram-negative pathogens suggested this process was proteolytic in nature so previous experiments were repeated with a single inoculum (10.sup.7 CFU/mL) of each A. baumannii clinical isolate in our bacterial library along with E. coli (strong reductase) and P. aeruginosa (known IgA protease) in the presence or absence of protease inhibitor. Although a reduction of SIgA degradation by P. aeruginosa was seen following incubation with protease inhibitor, no difference was observed with any of the other strains tested suggesting A. baumannii utilized a process that was not proteolytic in nature to break down SIgA FIG. 1B. Next, two competitive inhibitors of reductase enzymes targeting the thioredoxin fold (-C-X-X-C-) motif typically found in thiol-reductase enzymes (thioredoxin, glutathione, thioredoxin reductase, etc) were identified. The first, dithionitrobenzoic acid (Ellman's reagent) is often used as a colorimetric substrate to monitor thioredoxin activity as it produces a bright yellow color upon cleavage of the disulfide bond within the molecule. The second is PX-12. Both of these inhibitors completely ablated SIgA reduction and SC liberation 2 hours after incubation at concentrations of 1 mM and 15 g/mL, respectively (FIG. 1C). Although not shown here, inhibition was still evident at 24 hours with Ellman's reagent, PX-12 inhibition seemed to be overcome by the bacteria after about 12 hours. This may suggest that either the bacteria are producing more reductase enzyme eventually overwhelming the concentrations of PX-12 in the mixture, or the observed PX-12 inhibition is not irreversible with respect to the bacterial reductase. (Error bars represent SEM in all graphs; statistical differences determined by ANOVA with Dunnett correction; * significance p<0.01; ** p<0.001).

[0072] FIG. 2 shows IgA enhances virulence of Acinetobacter baumannii during GI challenge. Wild type (WT) and IgA deficient (IgA.sup./) C57BL/6 mice were challenged with 510.sup.7 CFU by oral gavage and monitored for morbidity and mortality over the course of a month. 71% of WT mice challenged succumbed to infection compared to only 43% of IgA.sup./ mice indicating IgA is necessary for virulence of Acinetobacter baumannii during GI challenge (data representative of three independent experiments).

[0073] FIG. 3 shows IgA enhances Acinetobacter baumannii adherence and colonization in the GI Tract. Acinetobacter baumannii was stained with the cationic dye PSVue-794, a near-infrared fluorescent dye, and used to track progression of Acinetobacter baumannii through the GI tract following oral gavage. Wild type (WT), IgA deficient (IgA.sup./), and B-cell deficient (MT) mice were challenged with 510.sup.7 CFU of the stained bacteria and monitored over the course of two days by full body in vivo live imaging showing prolonged fluorescence in WT mice at all observation points (A). Region of interest (ROI) analysis confirmed these visual differences with significant differences (p<0.001) in mean fluorescence intensities associated with WT mice compared to IgA.sup./ and MT mice at all observation points (B). Intestinal sections obtained from infant mice also show significantly (p<0.001) reduced bacterial adherence in sections from IgA.sup./ mice compared to WT (C).

[0074] FIG. 4 is a working model of Acinetobacter baumannii gastrointestinal infection. Dimeric IgA is produced by IgA secreting plasma cells at the lamina propria of the intestinal epithelium. The polymeric immunoglobulin receptor (pIgR) has high affinity for the J-chain of polymeric immunoglobulins and binds covalently to dimeric IgA. Binding facilitates translocation of the immunoglobulin to the intestinal lumen where pIgR is cleaved and released forming SIgA. During Acinetobacter baumannii infection, the bacteria reduce the bonds between the dimeric IgA and SC (the remnant of pIgR still bound to IgA) to form free SC. The unprotected IgA then becomes susceptible to degradation by digestive enzymes while free SC binds to Acinetobacter baumannii in a non-specific manner to form immune complexes with the bacteria. These complexes allow the non-motile bacterium to anchor itself within the mucous lining. In so doing, the bacteria can colonize and form biofilms within the gastrointestinal tract. Biofilm formation at the luminal surface of the enterocyte may result in increased TLR activation to stimulate NF-kB activation resulting in increased pro-inflammatory cytokine release and pIgR expression at the basal surface of the cell to allow for increased SIgA. Additionally, the immune complexes formed may be transcytosed by intestinal M-cells for presentation to dendritic cells (DC). If the bacteria are present in high enough numbers, bacterial outer membrane proteins may cause apoptosis in the DCs allowing the bacteria to disseminate into the body.

[0075] FIG. 5 shows SIgA breakdown and intestinal adhesion by Acinetobacter baumannii is inhibited by thioredoxin Inhibitor PX-12. SIgA breakdown by Acinetobacter baumannii is significantly reduced up to 2 hours following treatment with 18 g/mL PX-12 (A). This same dose significantly inhibits bacterial adherence to intestinal sections obtained from WT C57BL/6 mice (B).

[0076] FIG. 6 shows the antimicrobial effect of PX-12 on Acinetobacter baumannii. Initial experiments with PX-12 examining the effect of PX-12 on SIgA reduction by A. buamannii revealed unexpected results with respect to bacterial growth. PX-12 concentrations used in these experiments approximated the concentration reported well tolerated by mice (500 g/mouse; 500 g/mL for this experiment). This dose resulted in a visible decrease in bacterial pellet size when supernatant was collected for analysis. After performing a minimum inhibitor concentration (MIC) determination, the inventors discovered that the MIC for PX-12 with respect to the multi-drug resistant (MDR) Acinetobacter baumanniii clinical isolates, with one exception, was 31.25 g/mL. Furthermore, this concentration was also bactericidal leading us to believe it may potentially be useful as a new antimicrobial compound for treating MDR Acinetobacter baumannii. (Bacterial concentrations used in presented studies 100 higher than recommended by CLSI standards)

[0077] FIG. 7 shows sections of small intestine (mostly duodenal/ileal) from infant WT, IgA.sup./, pIgR.sup./, and MT mice. Intestinal sections were cut down one side to expose the inner lumen of the intestine and individually placed in suspensions of A. baumannii strain Ci79 (10.sup.7 CFU/mL). The sections were incubated in this mixture for 30 minutes, washed twice in 250 volumes of sterile PBS and soaked in 500 volumes of PBS (volume of section 50-100 L) for five minutes. The remaining bound bacteria were enumerated through homogenization of each section into single cell suspensions in 10 mL sterile PBS followed by dilution plating. Using the intestinal sections collected from WT mice as the 100% control, nearly 80% reductions in bacterial adherence were observed in IgA deficient intestinal sections obtained from IgA.sup./, pIgR.sup./, and MT mice compared to WT. Additionally, treatment with the mammalian thioredoxin-1 inhibitor PX-12 also significantly reduced bacterial attachment by 40%.

[0078] FIG. 8 shows thioredoxin as a mediator of SIgA breakdown. Although inhibition of SIgA reduction was observed with compounds such as dithionitrobenzoic acid and PX-12, both known substrates of thiol-reducing enzymes, there are many A. baumannii enzymes classified as reductases. In order to narrow this list of potential mediators of SIgA reduction, RNA sequencing on A. baumannii strain Ci79 was performed. The transcriptome expression profile for A. baumannii treated for 1 hour with SIgA against untreated A. baumannii was assessed using Ion Torrent Personal Genomics Machine (PGM). Eighteen genes involved in reduction-oxidization reactions, based on gene ontology (GO) classifications, were identified and examined for fold change difference in gene expression following SIgA exposure. Of these 18 genes, only one exhibited a fold change greater than 2 following SIgA exposure. This gene, M212_3532, was annotated as thioredoxin-A (trxA), the bacterial homologue to mammalian thioredoxin-1. Although, not as greatly modulated, M212_0650, also annotated as trxA, exhibited increased gene expression (>1). Based on these data, trxA gene expression was examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) over time. Significantly increased gene expression of trxA (4.5 fold) 2 hours after exposure to SIgA by the 2.sup.Ct method was observed (FIG. 8B). Subsequently gene sequences corresponding to trxA in 34 Acinetobacter spp. isolates were extracted and assessed for phylogenetic relatedness by PhymL following ClustalW alignment utilizing Geneious analysis software. The resulting phylogenetic tree with corresponding bootstrap values indicated a very high level of genetic conservation between Acinetobacter spp. with respect to the thioredoxin-A gene sequence (FIG. 8C). In fact, within the Baumannii clade there was nearly 100% sequence homology between strains. Other clades include Calcoaceticus, and non-A. baumannii-calcoaceticus (non-ABC). (Error bars represent SEM (C); statistical difference determined by Welch t-test; * significance p<0.01)

[0079] FIG. 9 shows reduction of SIgA by recombinant A. baumannii thioredoxin-A (abTrxA). Recombinant abTrxA derived from A. baumannii clinical isolate Ci79 was expressed in Rosetta E. coli cells and purified using an amylose resin column. Following elution of the protein with maltose, E. coli derived thioredoxin reductase (ecTrxB) was found to have eluted with the purified protein. As a result, although reduction of SIgA was observed in the absence of NADPH (left), reduction of SIgA was enhanced with addition of 400 M NADPH (center). NADPH had no effect on SIgA in the absence of recombinant protein (right). This pattern of SIgA reduction was identical to that observed with bacteria alone.

II. Attenuated Acinetobacter baumannii and Vaccines

[0080] The inventors have created a new attenuated Acinetobacter baumannii mutant from a multi-drug resistant clinical isolate deficient in thioredoxin-A (TrxA). The inventors have discovered that the attenuated Acinetobacter baumannii possess decreased virulence and can be used in a vaccine to prevent Acinetobacter baumannii infection. The vaccine can be administered as a vaccine and/or in conjunction with the administration of an antimicrobial agent, such as the ones described herein or other known in the art. The vaccine can be administered to a subject orally, parenterally, by inhalation spray, nebulizer, topically, rectally, nasally, buccally, etc. The inventors have discovered that live attenuated Acinetobacter baumannii can be used to vaccinate a subject.

[0081] FIG. 11 shows a non-limiting example of a method for producing attenuated Acinetobacter baumannii deficient in TrxA (trxA). pGEM-T Easy lacks the necessary origin for Acinetobacter replication. In this example, cryotransformation was used and trxA bacteria were screened by erthyromycin resistance conveyed by the erm.sup.r gene used to replace TrxA.

[0082] FIG. 12 shows that the virulence of attenuated Acinetobacter baumannii is decreased in comparison to wild type (WT) strains in mice in a sepsis challenge orally dosed with either WT or trxA. trxA doses of 210.sup.7 and less showed no virulence in this assay.

[0083] FIG. 13 shows that surviving mice from the sepsis challenge described in FIG. 11 showed increased survival rates in a secondary sepsis challenge using an otherwise lethal dose of Acinetobacter baumannii than mice not previously exposed to any form of Acinetobacter baumannii (nave).

[0084] FIG. 14 shows an i.p. vaccination sepsis model used to test the effectiveness of live attenuated Acinetobacter baumannii as a vaccine against an otherwise lethal dose of Acinetobacter baumannii (WT Ci79 at 810.sup.5 CFU/mouse (1.6 LD.sub.50).

[0085] FIG. 15 shows the percent survival of mice in the primary sepsis challenge described in the FIG. 14 methodology. The virulence of attenuated Acinetobacter baumannii is decreased in comparison to wild type (WT) strains. Again, trxA doses of 210.sup.7 and less showed no virulence in this assay.

[0086] FIG. 16 shows Ci79 specific and non-specific humoral responses to the primary challenge described in FIG. 14.

[0087] FIG. 17 shows the percent survival of mice that survived the primary sepsis challenge in a secondary sepsis challenge as described in FIG. 14. Increased survival rates were seen in the primary sepsis challenge surviving mice compared to mice not previously exposed to any form of Acinetobacter baumannii (nave).

[0088] FIG. 18 shows total Ig bound to Ci79 coated plates from blood taken from the mice tested in the secondary challenge described in the FIG. 14 methodology.

[0089] FIG. 19 shows total Ig bound to TrxA coated plates from blood taken from the mice tested in the secondary challenge described in the FIG. 14 methodology.

[0090] FIG. 20 shows methodology for testing Ig against Ci79 over time after a primary inoculation of mice with Acinetobacter baumannii and attenuated Acinetobacter baumannii and to test survival of mice exposed to a secondary Acinetobacter baumannii sepsis challenge using a 100% LD.sub.50 amount of Acinetobacter baumannii (approximately 510.sup.7 CFU/mouse).

[0091] Certain embodiments are directed to a vaccine composition for prevention or treatment of bacterial infection. The compositions described herein can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical, pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), intravesical, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.

[0092] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, semisolids, monophasic compositions, multiphasic compositions (e.g., oil-in-water, water-in-oil), foams microsponges, liposomes, nanoemulsions, aerosol foams, polymers, fullerenes, and powders (see, e.g., Taglietti et al. (2008) Skin Ther. Lett. 13:6-8). Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0093] Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

[0094] Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carder compounds and other pharmaceutically acceptable carriers or excipients.

[0095] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

[0096] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0097] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0098] The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

[0099] The compositions of the present invention may include excipients known in the art. Examples of excipients used for vaccine formulation such as adjuvents, stabilizers, preservatives, and trace products derived from vaccine manufacturing processes include but are not limited to: Aluminum Hydroxide, Amino Acids, Benzethonium Chloride, Formaldehyde or Formalin, Inorganic Salts and Sugars, Vitamins, Asparagine, Citric Acid, Lactose, Glycerin, Iron Ammonium Citrate, Magnesium Sulfate, Potassium Phosphate, Aluminum Phosphate, Ammonium Sulfate, Casamino Acid, Dimethyl-betacyclodextrin, 2-Phenoxyethanol, Bovine Extract, Polysorbate 80, Aluminum Potassium Sulfate, Gelatin, Sodium Phosphate, Thimerosal, Sucrose, Bovine Protein, Lactalbumin Hydrolysate, Formaldehyde or Formalin, Monkey Kidney Tissue, Neomycin, Polymyxin B, Yeast Protein, Aluminum Hydroxyphosphate Sulfate, Dextrose, Mineral Salts, Sodium Borate, Soy Peptone, MRC-5 Cellular Protein, Neomycin Sulfate, Phosphate Buffers, Polysorbate, Bovine Albumin or Serum, DNA, Potassium Aluminum Sulfate, Amorphous Aluminum Hydroxyphosphate Sulfate, Carbohydrates, L-histidine, Beta-Propiolactone, Calcium Chloride, Neomycin, Ovalbumin, Potassium Chloride, Potassium Phosphate, Sodium Phosphate, Sodium Taurodeoxycholate, Egg Protein, Gentamicin, Hydrocortisone, Octoxynol-10, -Tocopheryl Hydrogen Succinate, Sodium Deoxycholate, Sodium Phosphate, Beta-Propiolactone, Polyoxyethylene 910, Nonyl Phenol (Triton N-101, Octoxynol 9), Octoxinol-9 (Triton X-100), Chick Kidney Cells, Egg Protein, Gentamicin Sulfate, Monosodium Glutamate, Sucrose Phosphate Glutamate Buffer Calf Serum Protein, Streptomycin, Mouse Serum Protein, Chick Embryo Fibroblasts, Human Albumin, Sorbitol, Sodium Phosphate Dibasic, Sodium Bicarbonate, Sorbitol, Sucrose, Potassium Phosphate Monobasic, Potassium Chloride, Potassium Phosphate Dibasic, Phenol, Phenol Red (Phenol sulfonphthalein), Amphotericin B, Chicken Protein, Chlortetracycline, Ethylenediamine-Tetraacetic Acid Sodium (EDTA), Potassium Glutamate, Cell Culture Media, Sodium Citrate, Sodium Phosphate Monobasic Monohydrate, Sodium Hydroxide, Calcium Carbonate, D-glucose, Dextran, Ferric (III) Nitrate, L-cystine, L-tyrosine, Magnesium Sulfate, Sodium Hydrogenocarbonate, Sodium Pyruvate, Xanthan, Peptone, Disodium Phosphate, Monosodium Phosphate, Polydimethylsilozone, Hexadecyltrimethylammonium Bromide Ascorbic Acid, Casein, Galactose, Magnesium Stearate, Mannitol, Hydrolyzed Porcine Gelatin, Freund's emulsified oil adjuvants (complete and incomplete), Arlacel A, Mineral oil, Emulsified peanut oil adjuvant (adjuvant 65), Corynebacterium granulosum-derived P40 component, Lipopolysaccharide, Mycobacterium and its components, Cholera toxin, Liposomes, Immunostimulating complexes (ISCOMs), Squalene, and Sodium Chloride.

[0100] Dosing may be dependent on severity and responsiveness of the condition or disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the condition or disease state is achieved, or until optimal immune response is achieved, or until optimal protection against future infection is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. The administering professional (e.g., physician) can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of the agent (e.g., molecule, oligonucleotide, siRNA, antibody, virus, microbe, cell, bacterial cell), and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models or based on the examples described herein. In general, dosage is from 0.01 g to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly. The administering professional can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the subject undergo maintenance therapy to prevent the recurrence of the disease state, wherein the treatment (e.g., molecule, siRNA or antibody, virus, microbe, cell, bacterial cell) is administered in maintenance doses, ranging from 0.01 g to 100 g per kg of body weight, once or more daily, to once every 20 years.

[0101] Embodiments of the present invention have been shown to act as a live attenuated vaccine for the prevention of infection. The present invention is not limited to a particular dose, administration route, or administration regime to a subject. The vaccine may be administered at least once; twice; three times; four times; 5-10 times; 10-20 times; 20-100 times. The method is not limited by the duration of time between each repetition of vaccine administration. The method is not limited by the duration of time between administration of the vaccine and challenge or exposure to a pathogenic agent. The duration of time may be 0 days; 1 day; 2 days; 3 days; 4 days; 5 days; 5-7 days; 1-2 weeks; 2-4 weeks; 4-8 weeks; 8-10 weeks; 10-31 weeks; 31-52 weeks; 1-5 years; 5-10 years; 10-20 years; 20-50 years; 50-100 years.

[0102] The term pharmaceutically acceptable carrier refers to a carrier that may be administered to a patient, together with a compound of this invention, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound.

III. Thioredoxin and Thioredoxin Inhibitors

[0103] Thioredoxin plays a role in promoting eukaryotic cell survival, proliferation, and tumor angiogenesis, which makes it an attractive molecular target for therapeutic intervention in cancer. PX-12 (1-methylpropyl 2-imidazolyl disulfide) is a thioredoxin inhibitor that irreversibly binds rendering thioredoxin redox inactive and is being investigated as a potential cancer treatment.

[0104] The thioredoxin superfamily of proteins is characterized by a motif known as a thioredoxin fold (-C-X-X-C-) which serves as the active site of the enzyme. One of the cysteine residues forms a disulfide bond with disulfide substrate, resulting in reduction and release of one half of the molecule (which can either be an organic molecule containing a disulfide linkage or sulfur containing amino acid of an adjacent protein). The second cysteine residue of the active site then forms a disulfide bond with the first resulting in reduction of the other half of the substrate. Substrates for the above enzymes, such as asymmetric disulfide compounds, of which include but are not limited to compounds such as the imidazole disulfide PX-12, act as a substrate to these enzymes and can consequently reversibly inhibit these enzymes in a competitive fashion. In the case of thioredoxin, specifically, a third cysteine residue, Cys.sup.73, involved in dimerization of the enzyme can also act upon imidazole disulfide compounds. Doing so, however, may result in irreversible modification of the cysteine residue and prevent dimerization of the enzyme, a process necessary to reduce the enzyme back into its active state.

[0105] In certain aspects the thioredoxin inhibitor is an asymmetric disulfide, such as but not limited to 2-(sec-Butyldisulfanyl)-1H-imidazole; 2-(sec-Butyldisulfanyl)thiazole; 2-(sec-Butyldisulfanyl)pyridine; 2-(sec-Butyldisulfanyl)-3H-imidazo[4,5-c]pyridine; 2-(sec-Butyldisulfanyl)benzo[d]thiazole; 2-(sec-Butyldisulfanyl)-6-fluorobenzo[d]thiazole; 2-(sec-Butyldisulfanyl)-6-chlorobenzo[d]thiazole; 2-(sec-Butyldisulfanyl)-6-iodobenzo[d]thiazole; 4-Bromo-2-(sec-butyldisulfanyl)benzo[d]thiazole; 5-Bromo-2-(sec-butyldisulfanyl)benzo[d]thiazole; 2-(sec-Butyldisulfanyl)-6-nitrobenzo[d]thiazole; 2-(Ethyldisulfanyl)-1H-benzo[d]imidazole; 2-(tert-Butyldisulfanyl)-1H-benzo[d]imidazole; 2-(sec-Butyldisulfanyl)-1H-benzo[d]imidazole; 2-(Isopropyldisulfanyl)-1H-benzo[d]imidazole; 2-(Cyclopentyldisulfanyl)-1H-benzo[d]imidazole; 2-(Cyclohexyldisulfanyl)-1H-benzo[d]imidazole; 2-(Cyclohexyldisulfanyl)benzo[d]thiazole; 2-(Cyclohexyldisulfanyl)benzo[d]oxazole; 2-(sec-Butyl di sulfanyl)-6-chloro-5-fluoro-1H-benzo[d]imidazole; 6-Chloro-2-(cyclohexyldisulfanyl)-5-fluoro-1H-benzo[d]imidazole; 2-(sec-Butyldisulfanyl)-5-nitro-1H-benzo[d]imidazole; 2-(Cyclohexyldisulfanyl)-5-nitro-1H-benzo[d]imidazole; 2-(Cyclohexyldisulfanyl)-5-ethoxy-1H-benzo[d]imidazole; (2-(Cyclohexyldi sulfanyl)-1H-benzo[d]imidazol-6-yl)(phenyl)-methanone; 2-Amino-8-(cyclohexyldisulfanyl)-7H-purin-6-ol; 8-(Cyclohexyldisulfanyl)-7H-purin-6-amine; 2-(Cyclohexyldisulfanyl)-4H-benzo[d][1,3]thiazine; 2-(Cyclohexyldisulfanyl)-5-phenyl-1H-imidazole; or 3-(Cyclohexyldisulfanyl)-5-phenyl-4H-1,2,4-triazol-4-amine. In some aspects the thioredoxin inhibitor is 1-methylpropyl 2-imidazolyl disulfide (PX-12).

[0106] All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.