LIVE BACTERIAL VACCINES RESISTANT TO CARBON DIOXIDE (CO2), ACIDIC PH AND/OR OSMOLARITY FOR VIRAL INFECTION PROPHYLAXIS OR TREATMENT
20200023053 ยท 2020-01-23
Assignee
Inventors
Cpc classification
C12N7/00
CHEMISTRY; METALLURGY
C12N2760/16134
CHEMISTRY; METALLURGY
International classification
Abstract
Gram-negative bacterial mutants resistant to one or more stress conditions, including CO.sub.2, acid pH, and high osmolarity, and more particularly to gram-negative bacterial mutants with reduced TNF- induction having a mutation in one or more lipid biosynthesis genes, including, but not limited to msbB, that are rendered stress-resistant by a mutation in the zwf gene. Compositions are provided comprising one or more stress-resistant gram-negative bacterial mutants, preferably attenuated stress-resistant gram-negative bacterial mutants. Methods are provided for prophylaxis or treatment of a virally induced disease in a subject comprising administering to a subject a stress-resistant gram-negative bacterial mutant, preferably attenuated stress-resistant gram-negative bacterial mutants. The stress-resistant gram-negative bacterial mutants may serve as vectors for the delivery of one or more therapeutic molecules to a host. The methods of the invention provide more efficient delivery of therapeutic molecules by stress-resistant gram-negative bacterial mutants engineered to express said therapeutic molecules.
Claims
1. A method of treating a human or animal, comprising: administering to the human or mammal a live genetically engineered bacteria derived from a wild type species having an MsbB gene and a zwf gene, the live genetically engineered bacteria having a knockout mutation of MsbB and a knockout mutation of zwf; allowing the live genetically engineered bacteria to replicate within and colonize a tissue of the human or animal having a pH of pH 6.7 or below, to cause a transient maintenance of the live genetically engineered bacteria in the tissue; and secreting, by the live genetically engineered bacteria, within the tissue, a heterologous protein.
2. The method according to claim 1, further comprising clearing the live genetically engineered bacteria from the tissue.
3. The method according to claim 1, wherein the heterologous protein comprises an antigen adapted act as a vaccine.
4. The method according to claim 1, wherein the heterologous protein comprises a eukaryotic-type antigen adapted act as a vaccine.
5. The method according to claim 1, wherein the heterologous protein comprises a fusion of a bacterial-type secretion signal and an antigenic peptide portion.
6. The method according to claim 1, wherein the live genetically engineered bacteria are Salmonella.
7. The method according to claim 1, wherein the wild type species is Salmonella enterica.
8. The method according to claim 1, wherein the live genetically engineered bacteria are zwf Salmonella YS1646 ATCC Accession No. 202165.
9. The method according to claim 1, wherein the live genetically engineered bacteria have at least one mutation in a biosynthetic pathway selected from the group consisting of the isoleucine biosynthetic pathway, valine biosynthetic pathway, phenylalanine biosynthetic pathway, tryptophan biosynthetic pathway, tyrosine biosynthetic pathway, and arginine biosynthetic pathway.
10. A live genetically engineered bacteria derived from a wild type species having an MsbB gene and a zwf gene, comprising: a knockout mutation of MsbB; and a knockout mutation of zwf; at least one gene configured to cause secretion of a heterologous protein; the live genetically engineered bacteria being adapted to replicate within and colonize a tissue of a human or animal having a pH of pH 6.7 or below, to cause a transient maintenance of the live genetically engineered bacteria in the tissue.
11. The live genetically engineered bacteria according to claim 10, wherein the wild type species is Salmonella, and live genetically engineered bacteria is adapted to colonize a gut of a human recipient of the live genetically engineered bacteria.
12. The live genetically engineered bacteria according to claim 10, wherein the heterologous protein comprises an antigen adapted to induce a protective vaccination immune response of the human or animal.
13. The live genetically engineered bacteria according to claim 10, wherein the heterologous protein comprises an antigen adapted to induce a therapeutic immune response of the human or animal.
14. The live genetically engineered bacteria according to claim 10, wherein the live genetically engineered bacteria, are adapted after colonization, to produce a therapeutically effective amount of the heterologous protein.
15. The live genetically engineered bacteria according to claim 10, wherein the live genetically engineered bacteria, are adapted after colonization, to produce a therapeutically effective amount of the heterologous protein to induce an immune response against an infectious organism.
16. The live genetically engineered bacteria according to claim 10, wherein the heterologous protein comprises a fusion protein having at least a bacterial secretion signal and a eukaryotic-type antigenic peptide.
17. The live genetically engineered bacteria according to claim 10, in combination with a pharmaceutically acceptable carrier.
18. A live genetically engineered bacterium, comprising: a knockout mutation of MsbB; and a knockout mutation of zwf; the knockout mutation of MsbB and the knockout mutation of zwf together causing the live genetically engineered bacterium to have resistance to growth suppressive effects of CO.sub.25%, pH6.7, and osmolarity of 455 milliosmoles; at least one gene configured to cause secretion from the live genetically engineered bacterium of a heterologous protein; the live genetically engineered bacteria being adapted to replicate within and colonize a tissue of a human or animal having a pH of pH 6.7 or below, to cause a transient maintenance of the live genetically engineered bacteria in the tissue.
19. The live genetically engineered bacterium according to claim 18, further comprising: an attenuating mutation of at least one gene to auxotrophy; and the heterologous protein comprises a fusion of a therapeutic peptide portion and a secretion signal.
20. The live genetically engineered bacteria according to claim 19, wherein the therapeutic peptide sequence portion comprises a eukaryotic protein antigen.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0107] The invention provides gram-negative bacterial mutants resistant to one or more stress conditions, including, but not limited to, CO.sub.2, acid pH, and/or high osmolarity. In one embodiment, the present invention provides gram-negative bacterial mutants resistant to CO.sub.2, acid pH, and/or high osmolarity. In a more preferred embodiment, the present invention provides attenuated gram-negative bacterial mutants resistant to CO.sub.2, acid pH, and/or high osmolarity. Preferably, the stress-resistant gram-negative bacterial mutants are attenuated by introducing one or more mutations in one or more genes in the lipopolysaccharide (LPS) biosynthetic pathway that reduces the induction of TNF-, and optionally, one or more mutations to auxotrophy for one or more nutrients or metabolites.
[0108] The invention also provides stress-resistant gram-negative bacterial mutants engineered to contain and/or express one or more nucleic acid molecules encoding one or more therapeutic molecules. In a specific embodiment, the present invention provides stress-resistant gram-negative mutants engineered to contain and/or express one or more nucleic acid molecules encoding one or more therapeutic molecules. In another embodiment, the present invention provides attenuated stress-resistant gram-negative mutants engineered to contain and/or express one or more nucleic acid molecules encoding one or more therapeutic molecules. In yet another preferred embodiment, the present invention provides attenuated stress-resistant gram-negative mutants engineered to contain and/or express one or more nucleic acid molecules encoding one or more therapeutic molecules.
[0109] The invention also provides pharmaceutical compositions comprising pharmaceutically acceptable carriers and one or more stress-resistant gram-negative bacterial mutants, preferably one or more stress-resistant gram-negative bacterial mutants. The invention also provides pharmaceutical compositions comprising pharmaceutically acceptable carriers and one or more stress-resistant gram-negative bacterial mutants, comprising nucleotide sequences encoding one or more therapeutic molecules. The pharmaceutical compositions of the invention may be used in accordance with the methods of the invention for prophylaxis or treatment of virally-induced disease. Preferably, the stress-resistant gram-negative bacterial mutants are attenuated by introducing one or more mutations in one or more genes in the lipopolysaccharide (LPS) biosynthetic pathway, and optionally one or more mutations to auxotrophy for one or more nutrients or metabolites.
[0110] The present invention encompasses treatment protocols that provide a better therapeutic effect than current existing vaccines. In particular, the present invention provides methods for prevention or treatment of virally-induced disease in a subject comprising administering to said subject and one or more stress-resistant gram-negative bacterial mutants. The present invention also provides methods for the for viral infection prophylaxis or treatment in a subject comprising administering to said subject one or more stress-resistant gram-negative bacterial mutants, preferably attenuated stress-resistant gram-negative bacterial mutants, wherein said stress-resistant gram-negative bacterial mutants comprise one or more nucleic acid molecules encoding one or more therapeutic molecules.
[0111] The present invention provides methods for the enhanced delivery of one or more therapeutic molecules for prophylaxis and treatment of virally-induced disease comprising administering to said subject one or more stress-resistant gram-negative bacterial mutants, comprising nucleic acid molecules encoding one or more therapeutic molecules. The methods of the present invention permit lower dosages and/or less frequent dosing of stress-resistant gram-negative bacterial mutants (preferably attenuated stress-resistant gram-negative bacterial mutants) to be administered to a subject for prophylaxis or treatment of virally-induced disease to achieve a therapeutically effective amount of one or more therapeutic molecules.
[0112] The invention also provides a pharmaceutical pack or kit comprising one or more containers with one or more of the components of the pharmaceutical compositions of the invention. The kit further comprises instructions for use of the composition(s). In certain embodiments of the invention, the kit comprises a document providing instructions for the use of the composition(s) of the invention in, e.g., written and/or electronic form. Said instructions provide information relating to, e.g., dosage, methods of administration, and duration of treatment. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products which notice reflects approval by the agency of manufacture, use or sale for human administration.
[0113] For reasons of clarity, the detailed description is divided into the following subsections: Stress-Resistant Gram-Negative Bacterial Mutants; Production of Stress-Resistant Gram-Negative Bacterial Mutants; Identification and Selection of Stress-Resistant Gram-Negative Bacterial Mutants; Genetic Modifications to Stress-Resistant Mutants with Transposon Insertions or Multicopy Plasmids; Therapeutic Molecules; Expression Vehicles Methods and Compositions for Delivery; Methods of Determining the Therapeutic Utility; and Kits.
[0114] Stress-Resistant Gram-Negative Bacterial Mutants
[0115] Any gram-negative bacterial with the ability to grow under one or more environmental stresses such as those that exist in animals (i.e., stress-resistant gram-negative bacterial mutants) may be used in the compositions and methods of the invention. Examples of environmental stresses include, but are not limited to, CO.sub.2 resistant, acid pH resistant, and/or osmolarity resistant gram-negative bacterial mutants (methods for identifying, isolating, and producing such gram-negative bacterial are described infra). In a specific embodiment, the gram-negative bacteria used in the compositions and methods of the invention are CO.sub.2 resistant and/or acid pH resistant gram-negative bacterial mutants.
[0116] In a preferred embodiment, the stress-resistant gram-negative bacterial mutants used in the compositions and methods of the invention are attenuated. Any technique well-known to one of skill in the art may be used to attenuate the stress-resistant gram-negative bacterial mutants. Preferably, the stress-resistant gram-negative bacterial mutants used in the compositions and methods of the invention are attenuated by the introduction of one or more mutations in one or more genes in the lipopolysaccharide (LPS) biosynthetic pathway, and optionally, one or more mutations to auxotrophy for one or more nutrients or metabolites. Examples of genes in the LPS biosynthetic pathway which may be attenuated include, but are not limited to, htrB, msbB, kdsA, kdsB, kdtA, lpxB, lpxC, and lpxD. Mutations to auxotrophy can be produced by the introduction of one or more mutations in a gene in a biosynthetic pathway such as the leucine, isoleucine, valine, phenylalanine, tryptophan, tyrosine, arginine, uracil, or purine biosynthetic pathway. In particular, a mutation in the AroA gene can result in auxotrophy. The attenuated stress-resistant gram-negative bacterial mutants induce lower levels of tumor necrosis factor- (TNF-) than their wild-type counterpart (i.e., about 5% to about 40%, about 5% to about 35%, about 5% to about 25%, about 5% to about 15%, or about 5% to about 10% of TNF- induced by wild-type) as measured by techniques well-known in the art (e.g., immunoassays such as ELISAs), and thus, avoid or reduce the risk of inducing septic shock in a subject administered a mutant bacterium for viral infection prophylaxis or treatment to said subject in accordance with the methods of the invention.
[0117] In a preferred embodiment, the stress-resistant gram-negative bacterial mutants used in the compositions and methods of the invention induce an immune response to avian influenza. The present invention encompasses compositions and methods for prophylaxis and treatment of virally-induced disease using stress-resistant gram-negative bacterial mutant which replicates at physiological temperatures (i.e., 35 C. to 44 C.) and induce an immune response in vitro or in vivo. Preferably, such bacteria inhibit or reduce viral burden in vivo. In accordance with the invention, such a gram-negative bacterial mutant may be engineered to contain or express one or more therapeutic molecules which have an anti-viral immunostimulatory activity in vivo.
[0118] While the teachings in sections of this application may refer specifically to Salmonella, the compositions and methods of the invention are in no way meant to be restricted to Salmonella but encompass any other gram-negative bacterium to which the teachings apply. Suitable bacteria which may be used in accordance with the invention include, but are not limited to, Escherichia coli including enteroinvasive Escherichia coli (e.g., enteroinvasive Escherichia coli), Shigella sp., and Yersinia enterocohtica. Thus, the reference to Salmonella in this application is intended to serve as an illustration and the invention is not limited in scope to Salmonella.
[0119] The present invention encompasses the use of Salmonella with the ability to grow under one or more environmental stresses such as those that exist within the body of an animal (i.e., stress-resistant Salmonella mutants) in the compositions and methods of the invention. Examples of environmental stresses include, but are not limited to, CO.sub.2 concentration, temperature, pH, and osmolarity. Preferably, the Salmonella used in the compositions and methods of the invention are CO.sub.2 resistant, acid pH resistant, and/or osmolarity resistant gram-negative bacterial mutants (methods for identifying, isolating, and producing such Salmonella are described infra). In a specific embodiment, the Salmonella used in the compositions and methods of the invention are CO.sub.2 resistant and/or acid pH resistant gram-negative bacterial mutants.
[0120] In a preferred embodiment, the stress-resistant Salmonella used in the methods and compositions of the invention are attenuated. Preferably, the attenuated stress-resistant Salmonella mutants used in the methods and compositions of the invention have one or more mutations in one or more genes which reduce the virulence and toxicity of Salmonella. In a preferred embodiment, the attenuated stress-resistant Salmonella used in the compositions and methods of the invention have mutation(s) in one or more genes in the lipopolysaccharide (LPS) biosynthetic pathway (preferably in the msbB gene) and optionally, have one or more mutations to auxotrophy for one or more nutrients or metabolites, such as uracil biosynthesis, purine biosynthesis, tyrosine biosynthesis, leucine, isoleucine biosynthesis, arginine biosynthesis, valine biosynthesis, tryptophan biosynthesis and arginine biosynthesis.
[0121] The growth of an attenuated stress-resistant Salmonella used in accordance with the invention may be sensitive to a chelating agent such as, e.g., Ethylenediaminetetraacetic Acid (EDTA), Ethylene Glycol-bis (-aminoethyl Ether) N, N, N, N-Tetraacetic Acid (EGTA), or sodium citrate. For example, a chelating agent may inhibit the growth of an attenuated Salmonella for viral infection prophylaxis or treatment by about 90%, 95%, 99%, or 99.5% compared to the growth of a wild-type Salmonella used in accordance with the invention survive in macrophages at about 50% to about 30%, about 0% to about 10%, or about 10% to about 1% of the level of survival of a wild-type Salmonella sp.
[0122] The present invention provides a mutant Salmonella sp. for viral infection prophylaxis or treatment comprising a genetically modified msbB gene and a mutation characterized by increased growth when grown under CO.sub.2 conditions compared to the msbB mutant Salmonella designated YS1646 having ATCC Accession No. 202165. The present invention also provides a mutant Salmonella sp. for viral infection prophylaxis or treatment comprising a genetically modified msbB gene and a mutation characterized by increased growth when grown in acidified media compared to the msbff mutant Salmonella designed YS1646 having ATCC Accession No. 202165. The present invention further provides a mutant Salmonella sp. for viral infection prophylaxis or treatment comprising a genetically modified msbB gene and a mutation characterized by increased growth in media with high osmolarity compared to the msbff mutant Salmonella designed YS1646 having ATCC Accession No. 202165. Such mutant Salmonella sp. may further comprise one or more genetically modified genes to auxotrophy. In a preferred embodiment, the present invention provides Salmonella mutants comprising a genetically modified msbB gene and a genetically modified zwf gene.
[0123] Characteristics of CO.sub.2-Resistant Gram-Negative Bacterial Mutant
[0124] The primary characteristic of CO.sub.2-resistant gram-negative bacterial mutants is the enhanced percentage of their recovery on LB agar in CO.sub.2 relative to the parental strain of bacteria from which they were derived. In one embodiment, the percent recovery of CO.sub.2-resistant gram-negative mutants grown under CO.sub.2 conditions is approximately 2% to approximately 95%, approximately 2% to approximately 75%, approximately 2% to approximately 50%, approximately 2% to about 40%, approximately 2% to about 30%, approximately 2% to about 25%, approximately 2% to about 20% or about 2% to approximately 10% greater than the recovery of the parental strain of bacteria from which the CO.sub.2-resistant gram-negative bacterial mutants were derived grown under the same conditions.
[0125] A secondary characteristic of CO.sub.2-resistant gram-negative bacterial mutants with mutations in lipid biosynthesis genes that suppress TNF- induction is that the derived mutant retains the same low-level induction of TNF-. In one embodiment, the percent TNF- induction is approximately 2% to approximately 95%, approximately 2% to approximately 75%, approximately 2% to approximately 50%, approximately 2% to about 40%, approximately 2% to about 30%, approximately 2% to about 25%, approximately 2% to about 20% or about 2% to approximately 10% that of the wild type strain of bacteria grown under the same conditions.
[0126] As the pH tends to drop during incubation in 5% CO.sub.2, some CO.sub.2-resistant gram-negative mutants may have increased growth in acidified media relative to the parental strain of bacteria from which they were derived. Thus, CO.sub.2-resistant clones may be tested for resistance to acidic pH (such as pH 6.7 or lower), utilizing the methods described infra. In one embodiment, CO.sub.2-resistant gram-negative mutants grow approximately 2% to approximately 95%, approximately 2% to approximately 75%, approximately 2% to approximately 50%, approximately 2% to approximately 40%, approximately 2% to approximately 30%, approximately 2% to approximately 25%, or approximately 2% to approximately 10% less in acidified media than the parental strains of bacteria from the CO.sub.2-resistant gram-negative mutants were derived.
[0127] In addition, some CO.sub.2-resistant gram-negative mutants may be more attenuated than the parental strains of bacteria from which they were derived.
[0128] Characteristics of Acid pH-Resistant Gram-Negative Bacterial Mutants
[0129] The primary characteristic of acid pH-resistant gram-negative bacterial mutants is their ability to grow in liquid media under acidic pH conditions (e.g., pH 6.7, pH 6.6, pH 6.5, pH 6.25, pH 6.0, pH 5.5, pH 5.0, pH 4.5, pH 4.0, pH 3.5, pH 3.0, pH 2.5, pH 2.0, pH 1.5, pH 1.0 or lower) relative to the parental strain of bacteria from which they were derived. In one embodiment, the growth of the acid pH-resistant gram-negative mutants in acidified media is approximately 2% to approximately 95%, approximately 2% to approximately 75%, approximately 2% to approximately 50%, approximately 2% to approximately 40%, approximately 2% to approximately 30%, approximately 2% to approximately 20% or approximately 2% to approximately 10% or higher than the growth of the parental strain of bacteria from which the acid pH-resistant gram-negative bacterial mutants were derived grown under the same conditions. In a preferred embodiment, the growth of the acid pH-resistant gram-negative mutants in acidified media is approximately 40% to 100% higher than the growth of the parental strain of bacteria from which the acid pH-resistant gram-negative bacterial mutants were derived grown under the same conditions.
[0130] A secondary characteristic of acidic pH-resistant gram-negative bacterial mutants with mutations in lipid biosynthesis genes that suppress TNF- induction is that the derived mutant retains the same low-level induction of TNF-. In one embodiment, the percent TNF- induction is approximately 2% to approximately 95%, approximately 2% to approximately 75%, approximately 2% to approximately 50%, approximately 2% to about 40%, approximately 2% to about 30%, approximately 2% to about 25%, approximately 2% to about 20% or about 2% to approximately 10% that of the wild type strain of bacteria grown under the same conditions.
[0131] Some acid pH-resistant gram-negative mutants under CO.sub.2 conditions may have enhanced recovery relative to the parental strain of bacteria from which they were derived under the same conditions. Thus, acid pH-resistant clones may be tested for enhanced recovery under CO.sub.2 conditions, utilizing the methods described herein.
[0132] Some acid pH-resistant gram-negative mutants may have increased sensitivity to osmolarity. In addition, some acid pH-resistant gram-negative mutants may be more attenuated than the parental strains of bacteria from which they were derived.
[0133] Characteristics of Osmolarity-Resistant Gram-Negative Bacterial Mutants
[0134] The primary characteristic of osmolarity-resistant gram-negative bacterial mutants is their ability to survive and/or grow in media having high osmolarity relative to the parental strain of bacteria from which they were derived. In one embodiment, the survival and/or growth of the osmolarity-resistant gram-negative bacterial mutants in media having high osmolarity is approximately 2% to approximately 95%, approximately 2% to approximately 75%, approximately 2% to approximately 50%, approximately 2% to approximately 40%, approximately 2% to approximately 30%, approximately 2% to approximately 25%, approximately 2% to approximately 20% or approximately 2% to approximately 10% better than the survival and/or growth of the parental strain of bacteria from which the osmolarity-resistant gram-negative bacterial mutants were derived grown under the same conditions. In a preferred embodiment, the survival and/or growth of the osmolarity-resistant gram-negative bacterial mutants in media having high osmolarity is approximately 40% to 100% better than the survival and/or growth of the parental strain of bacteria from which the osmolarity-resistant gram-negative bacterial mutants were derived grown under the same conditions.
[0135] A secondary characteristic of osmolarity-resistant gram-negative bacterial mutants with mutations in lipid biosynthesis genes that suppress TNF- induction is that the derived mutant retains the same low-level induction of TNF-. In one embodiment, the percent TNF- induction is approximately 2% to approximately 95%, approximately 2% to approximately 75%, approximately 2% to approximately 50%, approximately 2% to about 40%, approximately 2% to about 30%, approximately 2% to about 25%, approximately 2% to about 20% or about 2% to approximately 10% that of the wild type strain of bacteria grown under the same conditions.
[0136] Some osmolarity-resistant gram-negative bacterial mutants may have increased sensitivity to CO.sub.2 and/or acid pH stress conditions relative to the parental strains of bacteria from which they were derived. Further, some osmolarity-resistant gram-negative bacterial mutants may have increased sensitivity to CO.sub.2 and/or acid pH stress conditions relative to the parental strains of bacteria from which they were derived, but the sensitivity of the osmolarity-resistant gram-negative bacterial mutants to CO.sub.2 and/or acid pH stress conditions is compensated for by other genetic alterations (e.g., alterations which cause resistance to CO.sub.2 and/or acid pH stress conditions).
[0137] In addition, some osmolarity-resistant gram-negative bacterial mutants may be more attenuated than the parental strains of bacteria from which they were derived.
[0138] Production of Stress-Resistant Gram-Negative Bacterial Mutants
[0139] Genetic alterations that confer resistance to one or more environmental stresses to gram-negative bacteria, preferably attenuated gram-negative bacteria and more preferably attenuated gram-negative bacteria for viral infection prophylaxis or treatment, can be produced utilizing any method well-known to one of skill in the art. For example, stress-resistant gram-negative bacterial mutants may be obtained by growing the bacteria under various selective pressures or by random mutagenesis (e.g., using a transposon library, using a multicopy plasmid library or by exposing the bacteria to various mutagens). Examples of growth condition parameters which may be varied to obtain stress-resistant mutants include, but are not limited to, the temperature, the type of media used to grow the bacteria, the pH of the media, and the CO.sub.2 concentration/levels. Examples of mutagens which may be used to obtain stress-resistant mutants include, but are not limited to, ultraviolet light and nitrosoguanadine.
[0140] Identification and Selection of Stress-Resistant Gram-Negative Bacterial Mutants
[0141] Gram-negative bacteria, preferably attenuated gram-negative bacteria, and/or preferably attenuated gram-negative bacteria for viral infection prophylaxis or treatment, with resistance to one or more environmental stresses can be identified and selected for utilizing any method well-known to one of skill in the art. In general, a pool of bacteria with genetic variations is subjected to one or more selection criteria and the resistant clones are isolated. A pool of gram-negative bacteria with genetic variations may be composed of spontaneous mutants, a library of transposon mutants or mutants transformed with a library of cloned DNA in a multicopy plasmid. The selected techniques that a pool of gram-negative bacteria with genetic variations is subjected to varies depending upon the particular stress-resistant mutant that one is attempting to select. Selection techniques for particular stress-resistant gram-negative bacteria may include, e.g., plating the bacteria to LB agar plates under the stress condition, growing the bacteria in LB broth under the stress condition, and then plating the bacteria to LB agar plates. Individual colonies are then isolated from the agar plates and tested for growth under the particular stress condition. Colonies with greater growth ability than the parental strain of bacteria from which they were derived are deemed to be resistant to the particular stress condition. In a specific embodiment, individual colonies of gram-negative bacteria with genetic variations are deemed to be resistant to a particular stress condition if they grow 2 fold, preferably 4 fold, 6 fold, 8 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 45 fold, 50 fold, 55 fold, 60 fold, 65 fold, 70 fold, 75 fold or higher levels under the stress condition than the parental strain of bacteria from which they were derived. In another embodiment, individual colonies of gram-negative bacteria with genetic variations are deemed to have resistance to a particular stress condition if their growth is 5%, preferably 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater under stress conditions than the parental strain of bacteria from which they were derived.
[0142] In order to distinguish spontaneous stress-resistant mutants from transposon and plasmid-based clones, the transposon or plasmid can be transferred to another strain using selection for the appropriate antibiotic marker found on the transposon or plasmid. Several sibling colonies may then be isolated and tested for resistance to the particular stress condition, thus avoiding spontaneous mutants. In order to simultaneously transfer large numbers of transposon or plasmid-based stress-resistant clones to distinguish them from spontaneous stress-resistant clones, following the first selection where the bacteria are grown under stress conditions, the clones may be pooled together, the genetic marker transferred, and then multiple sibling clones tested for growth under the stress condition.
[0143] Identification and Selection of CO.sub.2-Resistant Gram-Negative Bacterial Mutants
[0144] Gram-negative bacteria, preferably attenuated gram-negative bacteria, with resistance to CO.sub.2 can be identified and selected for utilizing any method well-known to one of skill in the art. In general, a pool of bacteria with genetic variations is subjected to one or more selection criteria and the resistant clones are isolated. A pool of gram-negative bacteria with genetic variations may be composed of spontaneous mutants, a library of transposon mutants or mutants transformed with a library of cloned DNA in a multicopy plasmid. Selection techniques for isolating CO.sub.2-resistant gram-negative bacteria may include, but are not limited to, growing the bacteria on LB agar plates at 37 C. under CO.sub.2 conditions, growing the bacteria in LB broth at 37 C. in CO.sub.2, and then growing the bacteria on LB agar plates at 37 C. under CO.sub.2 conditions or air. Individual colonies are then isolated from the agar plates and tested for plating efficiency on LB agar at 37 C. in air and LB agar at 37 C. in CO.sub.2. Colonies with greater than 0.5%, preferably 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% recover on the LB agar at 37 C. in CO.sub.2 are deemed to have enhanced resistance to CO.sub.2 relative to the parental strain of bacteria from which they were derived.
[0145] In order to distinguish spontaneous CO.sub.2-resistant mutants from transposon and plasmid-based clones, the transposon or plasmid can be transferred to another strain using selection for the appropriate antibiotic marker found on the transposon or plasmid. Several sibling colonies may then be isolated and tested for resistance to CO.sub.2, thus avoiding spontaneous mutants. In order to simultaneously transfer large numbers of transposon or plasmid-based stress-resistant clones to distinguish them from spontaneous CO.sub.2-resistant clones, following the first selection where the bacteria are grown on LB agar plates under CO.sub.2 conditions, the clones may be pooled together, the genetic marker transferred, and then multiple sibling clones tested for growth under the CO.sub.2 conditions.
[0146] Identification and Selection of Acid pH-Resistant Gram-Negative Bacterial Mutants
[0147] Gram-negative bacteria, preferably attenuated gram-negative bacteria, with resistance to acidic pH can be identified and selected for utilizing any method well-known to one of skill in the art. In general, a pool of bacteria with genetic variations is subjected to one or more selection criteria and the resistant clones are isolated. A pool of gram-negative bacteria with genetic variations may be composed of spontaneous mutants, a library of transposon mutants or mutants transformed with a library of cloned DNA in a multicopy plasmid. Selection techniques for isolating acid pH-resistant gram-negative bacteria may include, but are not limited to, plating the bacteria on LB agar plates at 37 C. at an acidic pH (e.g., pH 6.7, pH 6.6, pH 6.5, pH 6.4, pH 6.0, pH 5.5, pH 5.0, pH 4.5, pH 4.0, pH 3.5, pH 2.0, pH 2.5 or pH 1.0), growing the bacteria in LB broth at 37 C. at an acidic pH (e.g., pH 6.7, pH 6.6, pH 6.5, pH 6.4, pH 6.0, pH 5.5, pH 5.0, pH 4.5, pH 4.0, pH 3.5, pH 2.0, pH 2.5 or pH 1.0), and then plating the bacteria on LB agar plates at 37 C. Individual colonies are then isolated from the agar plates and tested for growth in acidified media at 37 C. Colonies with greater growth ability than the parental strain of bacteria from which they were derived are deemed to be resistant to a particular acid pH (e.g., pH 6.5, pH 6, pH 5, pH 4.5, pH 4, pH 3.5, pH 2, pH 2.5 or pH 1). In a specific embodiment, individual colonies of gram-negative bacteria with genetic variations are deemed to be resistant to an acidic pH (e.g., pH 6.5, pH 6, pH 5, pH 4.5, pH 4, pH 3.5, pH 2, pH 2.5 or pH 1) if they grow 2 fold, preferably 4 fold, 6 fold, 8 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 45 fold, 50 fold, 55 fold, 60 fold, 65 fold, 70 fold, 75 fold or higher levels in acidified media than the parental strain of bacteria from which they were derived. In another embodiment, individual colonies of gram-negative bacteria with genetic variations are deemed to have resistance to an acidic pH if their growth is 5%, preferably 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater in acidified media than the parental strain of bacteria from which they were derived.
[0148] In order to distinguish spontaneous acid pH-resistant mutants from transposon and plasmid-based clones, the transposon or plasmid can be transferred to another strain using selection for the appropriate antibiotic marker found on the transposon or plasmid. Several sibling colonies may then be isolated and tested for resistance to an acidic pH, thus avoiding spontaneous mutants. In order to simultaneously transfer large numbers of transposon or plasmid-based stress-resistant clones to distinguish them from spontaneous acid pH-resistant clones, following the first selection where the bacteria are grown on LB agar plates at an acidic pH, the clones may be pooled together, the genetic marker transferred, and then multiple sibling clones tested for growth in acidified media.
[0149] Identification and Selection of Osmolarity-Resistant Gram-Negative Bacterial Mutants
[0150] Gram-negative bacteria, preferably attenuated gram-negative bacteria, and more preferably attenuated gram-negative bacteria, with resistance to high osmolarity can be identified and selected for utilizing any method well-known to one of skill in the art. In general, a pool of bacteria with genetic variations is subjected to one or more selection criteria and the resistant clones are isolated. A pool of gram-negative bacteria with genetic variations may be composed of spontaneous mutants, a library of transposon mutants or mutants transformed with a library of cloned DNA in a multicopy plasmid. Selection techniques for isolating high osmolarity-resistant gram-negative bacteria may include, but are not limited to, growing the bacteria on agar plates having high osmolarity at 37 C., growing the bacteria in nutrient broth having high osmolarity at 37 C., and then growing the bacteria on agar plates having or not having high osmolarity at 37 C. Examples of agents that result in high osmolarity include, but are not limited to, salts (e.g., NaCl or KCl) and sugars (e.g., sucrose or glucose). Individual colonies are then isolated from the agar plates and tested for growth in media having high osmolarity at 37 C. Colonies with greater growth ability than the parental strain of bacteria from which they were derived are deemed to have resistance to high osmolarity. In a specific embodiment, individual colonies of gram-negative bacteria with genetic variations are deemed to have resistance to high osmolarity if they grow to 2 fold, preferably 4 fold, 6 fold, 8 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 45 fold, 50 fold, 55 fold, 60 fold, 65 fold, 70 fold, 75 fold or higher levels in media having high osmolarity than the parental strain of bacteria from which they were derived. In another embodiment, individual colonies of gram-negative bacteria with genetic variations are deemed to have resistance to high osmolarity if their growth is 5%, preferably 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater in media having high osmolarity than the parental strain of bacteria from which they were derived.
[0151] In order to distinguish spontaneous osmolarity-resistant mutants from transposon and plasmid-based clones, the transposon or plasmid can be transferred to another strain using selection for the appropriate antibiotic marker found on the transposon or plasmid. Several sibling colonies may then be isolated and tested for resistance to high osmolarity, thus avoid spontaneous mutants. In order to simultaneously transfer large numbers of transposon or plasmid-based stress-resistant clones to distinguish them from spontaneous osmolarity-resistant clones, following the first selection where the bacteria are grown on LB agar plates having high osmolarity, the clones may be pooled together, the genetic marker transferred, and then multiple sibling clones tested for growth in media having high osmolarity.
[0152] Genetic Modifications to Stress-Resistant Mutants with Transposon Insertions
[0153] Stress-resistant gram-negative mutants with transposon insertions can be re-engineered to have a deletion and/or insertion in the same site in order to eliminate the antibiotic resistance and transposon element. First, the site of the transposon insertion is determined using standard techniques well-known to those skilled in the art. Such techniques include, e.g., cloning from chromosomal DNA based on selection for antibiotic resistance and sequencing of the adjacent region, using GenomeWalker (Clontech, Palo Alto, Calif.) or direct chromosomal sequencing (Qiagen, Valencia, Calif.). A deletion and/or insertion is then constructed using PCR to generate the two segments necessary for the use of the sucrase vector (Donnenberg and Kaper, 1991, Infection and Immunity 59: 4310-4317). A multiple cloning site can be engineered at the junction of the two segments used to create an insertion. The insertion can be non-coding DNA or coding DNA (e.g., a nucleotide sequence encoding a therapeutic molecule such as prodrug-converting enzyme).
[0154] The genetic modification of a spontaneous mutant may be identified using standard techniques well-known to one of skill in the art. One technique to identify the genetic modification(s) of a spontaneous mutant uses linkage to transposons, as described by Murray et al., 2001, J. Bacteriology 183: 5554-5561. Another technique to identify the genetic modification(s) of a spontaneous mutant is to generate a DNA library derived from the strain of interest in a low-copy or transposon vector and to select for resistance to a particular stress condition. The plasmid or transposon DNA is then sequenced as described above. Another technique to identify the genetic modification(s) of a spontaneous mutant is to use a Genechip approach. In the Genechip approach differences between the spontaneous mutant and the parental strain are identified. The spontaneous deletion, rearrangement, duplication or other form of mutation identified in the spontaneous mutant may then be re-engineered into a multicopy plasmid such as asd vector or a sucrase chromosomal vector as described above.
[0155] Kits
[0156] Similarly, the invention may also provide a kit comprising (a) a first container comprising a bacterial expression codon optimized antigen from a pathogenic avian influenza virus strain containing unique genetically engineered restriction sites contained within either a bacterial protein expression plasmid or a bacterial chromosomal protein expression vector and (b) a second container comprising bacterial vector(s) with one or more (e.g., fH1, fH2 or fH0) flagellar antigen(s) and/or various non-overlapping O-antigens. Component (a) will be modifiable to genetically match an emerging avian influenza virus using standard in vitro molecular techniques and can be combined with component (b) to generate one or more bacterial strains with defined flagellar antigens which constitute a live vaccine. The variation(s) in flagellar antigens provided by the kit provide for more than one live vaccine strain in which a first immunization (prime) using one strain may be followed at an appropriate time such as 2 to 4 weeks by a second immunization (boost) using a second strain with a different fH antigen or no fH antigen. The live vaccine compositions are suitable for oral administration to an individual to provide protection from avian influenza.
[0157] The invention also provides a pharmaceutical pack or kit comprising one or more containers with one or more of the components of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
[0158] In a specific embodiment of the invention, the kit comprises one or more stress-resistant gram-negative bacterial mutants and optionally means of administering the pharmaceutical compositions of the invention. The different stress-resistant gram-negative bacterial mutants may comprise nucleotide sequences encoding one or more therapeutic molecules. The kit may further comprise instructions for use of said stress-resistant gram-negative bacterial mutants. In certain embodiments of the invention, the kit comprises a document providing instruction for the use of the composition of the invention in, e.g., written and/or electronic form. Said instructions provide information relating to, e.g., dosage, method of administration, and duration of treatment.
[0159] In one embodiment, a kit of the invention comprises a stress-resistant gram-negative bacterial mutant in a vial and instructions for administering the stress-resistant gram-negative bacterial mutants for viral prophylaxis or treatment, wherein the stress-resistant gram-negative bacterial mutant is a facultative anaerobe or facultative aerobe. In accordance with this embodiment, the stress-resistant gram-negative bacterial mutant may be engineered to express one or more nucleic acid molecules encoding one or more therapeutic molecules. In another embodiment, a kit of the invention comprises an anti-viral agent contained in a first vial, a stress-resistant gram-negative bacterial mutant in a second vial, and instructions for administering the anti-viral agent and stress-resistant gram-negative bacterial mutant to a subject for viral infection prophylaxis or treatment. In accordance with this embodiment, stress-resistant gram-negative bacterial mutant may be engineered to express one or more nucleic acid molecules encoding one or more therapeutic molecules. Preferably, the stress-resistant gram-negative bacterial mutants included in the kits of the invention are stress-resistant gram-negative Salmonella mutants.
[0160] In another embodiment, a kit of the invention comprises an attenuated stress-resistant gram-negative bacterial mutant in a vial and instructions for administering the attenuated stress-resistant gram-negative bacterial mutant to a subject for viral infection prophylaxis or treatment, wherein the attenuated stress-resistant gram-negative bacterial mutant is a facultative anaerobe or facultative aerobe. In accordance with this embodiment, the attenuated stress-resistant gram-negative bacterial mutant may be engineered to express one or more nucleic acid molecules encoding one or more therapeutic molecules. In another embodiment, a kit of the invention comprises an anti-viral agent contained in a first vial, an attenuated stress-resistant gram-negative bacterial mutant contained in a second vial, and instructions for administering the anti-viral agent and attenuated stress-resistant gram-negative bacterial mutant to a subject for viral infection prophylaxis or treatment. In accordance with this embodiment, the attenuated stress-resistant gram-negative bacterial mutant may be engineered to express one or more nucleic acid molecules encoding one or more therapeutic molecules. Preferably, the attenuated stress-resistant gram-negative bacterial mutants included in the kits of the invention are attenuated stress-resistant gram-negative Salmonella mutants.
[0161] In another embodiment, a kit of the invention comprises a stress-resistant gram-negative bacterial mutant for viral infection prophylaxis or treatment in a vial and instructions for administering the stress-resistant gram-negative bacterial mutant to a subject, where the stress-resistant gram-negative bacterial mutant is a facultative anaerobe or facultative aerobe. In accordance with this embodiment, the stress-resistant gram-negative bacterial mutant may be engineered to express one or more nucleic acid molecules encoding one or more therapeutic molecules. In another embodiment, a kit of the invention comprises an anti-viral agent contained in a first vial, a stress-resistant gram-negative bacterial mutant contained in a second vial, and instructions for administering the anti-viral agent and stress-resistant gram-negative bacterial mutant to a subject with a for viral infection prophylaxis or treatment. In accordance with this embodiment, the stress-resistant gram-negative bacterial mutant may be engineered to express one or more nucleic acid molecules encoding one or more therapeutic molecules.
[0162] The present invention incorporates a combination of bacterial vector and protein expression technology which results in a unique vaccine which is rapidly constructed in response to emerging avian influenza and their highly pathogenic derivatives. The present invention is directed to the construction bacterially codon optimized avian and human influenza genes and their incorporation into a Salmonella strain for therapeutic use in the prevention of avian influenza and highly pathogenic derivatives. An antigen-expressing plasmid or chromosomal construct in the bacterial strains described herein may also contain one or more transcriptional terminators adjacent to the 3 end of a particular nucleotide sequence on the plasmid to prevent undesired transcription into another region of the plasmid or chromosome. Such transcription terminators thus serve to prevent transcription from extending into and potentially interfering with other critical plasmid functions, e.g., replication or gene expression. Examples of transcriptional terminators that may be used in the antigen-expressing plasmids described herein include, but are not limited to, the TI and T2 transcription terminators from 5S ribosomal RNA bacterial genes (see, e.g., FIGS. 1-5; Brosius and Holy, Proc. Natl. Acad. Sci. USA, 81: 6929-6933 (1984); Brosius, Gene, 27(2): 161-172 (1984); Orosz et al., Eur. J Biochem., 20 (3): 653-659 (1991)).
[0163] The mutations in an attenuated bacterial host strain may be generated by integrating a homologous recombination construct into the chromosome or the endogenous Salmonella virulence plasmid (Donnenberg and Kaper, 1991; Low et al. (Methods in Molecular Medicine, 2003)). In this system, a suicide plasmid is selected for integration into the chromosome by a first homologous recombination event, followed by a second homologous recombination event which results in stable integration into the chromosome. The antigen-expressing chromosomal integration constructs described herein comprise one or more nucleotide sequences that encode one or more polypeptides that, in turn, comprise one or more avian influenza antigens, such as the hemagglutinin and neuraminidase polypeptide antigens, or immunogenic portions thereof, from avian influenza virus and highly pathogenic derivatives. Such coding sequences are operably linked to a promoter of transcription that functions in a Salmonella bacterial strain even when such a bacterial strain is ingested, i.e., when a live vaccine composition described herein is administered orally to an individual. A variety of naturally occurring, recombinant, and semi-synthetic promoters are known to function in enteric bacteria, such as Escherichia coli and serovars of S. enterica (see, e.g., Dunstan et al., Infect. Immun., 67(10): 5133-5141 (1999)). Promoters (P) that are useful in the invention include, but are not limited to, well known and widely used promoters for gene expression such as the naturally occurring Plac of the lac operon and the semi-synthetic Ptrc (see, e.g., Amman et al., Gene, 25 (2-3): 167-178 (1983)) and Ptac (see, e.g., Aniann et al., Gene, 69(2): 301-315 (1988)), as well as PpagC (see, e.g., Hohmann et al., Proc. Natl. Acad. Sci. USA, 92. 2904-2908 (1995)), PpmrH (see, e.g., Gunn et al., Infect. Immun., 68: 6139-6146 (2000)), PpmrD (see, e.g., Roland et al., J Bacteriol., 176: 3589-3597 (1994)), PompC (see, e.g., Bullifent et al., Vacccine, 18: 2668-2676 (2000)), PnirB (see, e.g., Chatfield et al., Biotech. (NY), 10: 888-892 (1992)), PssrA (see, e.g., Lee et al., J Bacteriol. 182. 771-781 (2000)), PproU (see, e.g., Rajkumari and Gowrishankar, J Bacteriol., 183. 6543-6550 (2001)), Pdps (see, e.g., Marshall et al., Vaccine, 18: 1298-1306 (2000)), and PssaG (see, e.g., McKelvie et al., Vaccine, 22: 3243-3255 (2004)), Some promoters are known to be regulated promoters that require the presence of some kind of activator or inducer molecule in order to transcribe a coding sequence to which they are operably linked. However, some promoters may be regulated or inducible promoters in E. coli, but function as unregulated promoters in Salmonella. An example of such a promoter is the well-known trc promoter (Ptrc, see, e.g., Amman et al., Gene, 25(2-3): 167-178 (1983); Pharmacia-Upjohn). As with Plac and Ptac, Ptrc functions as an inducible promoter in Escherichia coli (e.g., using the inducer molecule isopropyl-p-D-1 8 thio-galactopyranoside, IPTG), however, in Salmonella bacteria having no Lad repressor, Ptrc is an efficient constitutive promoter that readily transcribes avian influenza antigen-containing polypeptide coding sequences present on antigen-expressing plasmids described herein. Accordingly, such a constitutive promoter does not depend on the presence of an activator or inducer molecule to express an antigen-containing polypeptide in a strain of Salmonella.
[0164] The avian influenza antigen-expressing chromosomal integration constructs which integrate into the live vaccine strains also contain an origin of replication (ori) that enables the precursor plasmids to be maintained as multiple copies in certain the bacterial cells which carry the lambda pir element. For the process of cloning DNA, a number of multi-copy plasmids that replicate in Salmonella bacteria are known in the art, as are various origins of replications for maintaining multiple copies of plasmids. Preferred origins of replications for use in the multi-copy antigen-expressing plasmids described herein include the origin of replication from the multi-copy plasmid pBR322 (pBR ori; see, e.g., Maniatis et al., In Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1982), pp. 479-487; Watson, Gene, 70: 399-403, 1988), the low copy origin of replication from pACYC177, and the origin of replication of pUC plasmids (pUC ori), such as found on plasmid pUC 1 8 (see, e.g., Yanish-Perron et al., Gene, 33: 103-119 (1985)). Owing to the high degree of genetic identity and homology, any serovar of S. enterica may be used as the bacterial host for a live vaccine composition for avian influenza, provided the necessary attenuating mutations and antigen-expressing plasmids as described herein are also employed. Accordingly, serovars of S. enterica that may be used in the invention include those selected from the group consisting of Salmonella enterica serovar Typhimurium (S. typhimurium), Salmonella montevideo, Salmonella enterica serovar Typhi (S. typhi), Salmonella enterica serovar Paratyphi B (S. paratyphi B), Salmonella enterica serovar Paratyphi C (S. paratyphi C), Salmonella enterica serovar Hadar (S. Hadar), Salmonella enterica serovar Enteriditis (S. enteriditis), Salmonella enterica serovar Kentucky (S. kentucky), Salmonella enterica serovar Infantis (S. infantis), Salmonella enterica serovar Pullorum (S. pullorum), Salmonella enterica serovar Gallinarum (S. gallinarum), Salmonella enterica serovar Muenchen (S. muenchen), Salmonella enterica serovar Anaturn (S. anatum), Salmonella enterica serovar Dublin (S. dublin), Salmonella enterica serovar Derby (S. derby), Salmonella enterica serovar Choleraesuis var. kunzendorf (S. cholerae kunzendorf), and Salmonella enterica serovar minnesota (S. minnesota).
[0165] The vaccine compositions described herein may be administered orally to an individual in any form that permits the Salmonella bacterial strain of the composition to remain alive and to persist in the gut for a time sufficient to elicit an immune response to one or more avian influenza antigens of avian influenza virus and highly pathogenic derivatives expressed in the Salmonella strain. For example, the live bacterial strains described herein may be administered in relatively simple buffer or saline solutions at physiologically acceptable pH and ion content. By physiologically acceptable is meant whatever is compatible with the normal functioning physiology of an individual who is to receive a live vaccine composition described herein. Preferably, bacterial strains described herein are suspended in otherwise sterile solutions of bicarbonate buffers, phosphate buffered saline (PBS), or physiological saline, that can be easily swallowed by most individuals. However, oral routes of administration may include not only swallowing from the mouth a liquid suspension or solid form comprising a live bacterial strain described herein, but also administration of a suspension of a bacterial strain through a nasal spray or pulmonary inhaler, a nasojejunal or gastrostomy tube, and rectal administration, e.g., by using a suppository comprising a live bacterial strain described herein to establish an infection by such bacterial strain in the lower intestinal tract of the alimentary canal. Accordingly, any of a variety of alternative modes and means may be employed to administer a vaccine composition described herein to the alimentary canal of an individual if the individual cannot swallow from the mouth.
[0166]
[0167]
[0168]
[0169]
[0170]
[0171]
[0172]
[0173]
[0174]
[0175] In order to more fully illustrate the invention, the following non-limiting examples are provided.
Example 1: Isolation and Identification of a Gene Involved in Resistance to CO.SUB.2., Acidic pH and/or Osmolarity
[0176] Isolation of CO.sub.2 Resistant Strains Using Transposon Libraries.
[0177] Throughout the procedures, msbB.sup.+ strains were grown in Luria-Bertani (LB) broth containing 10 g tryptone, 5 g yeast extract, 10 g NaCl, pH adjusted as indicated using either 1N NaOH or 1N HCl, or LB plates containing 1.5% agar at 372 C. msbB.sup. strains were grown in modified LB referred to as MSB media (msbB media), containing 10 g tryptone, 5 g yeast extract 2 mL 1N CaCl.sub.2 and 2 mL 1N MgSO.sub.4 per liter, adjusted to pH 7.0 to 7.6 using 1N NaOH, or in LB broth or LB plates lacking NaCl, referred to as LB-0. For transductions, LB lacking EGTA was used. For sucrose resolutions, LB lacking NaCl and containing 5% sucrose at 302 C. was used. Auxotrophic mutants are determined on minimal media 56 (M56): 0.037 M KH.sub.2PO.sub.4, 0.06 M Na.sub.2HPO.sub.4, 0.02% MgSO.sub.47H.sub.2O, 0.2% (NH.sub.4).sub.2SO.sub.4, 0.001% Ca (NO.sub.3).sub.2, 0.00005% FeSO.sub.47H.sub.2O, with a carbon source (e.g., glucose 0.1 to 0.3%) as sterile-filtered additive, and further supplemented with the appropriate nutrients, 0.1 mg/ml thiamine and 50 mg/ml each of adenine. Solid M56 media is made by preparing separate autoclaved 2 concentrates of the mineral salts and the agar, which are combined after sterilization. Media are also supplemented with antibiotics used as needed to select for resistance markers, including tetracycline (Sigma) at 4 mg/ml from a stock: 10 mg/ml in 70% ethanol stored in darkness at 20 C. or ampicillin at 100 mg/ml from a stock: 100 mg/ml in H.sub.2O, sterile filtered and stored at 20 C. The bacteria used are listed in Table 1.
TABLE-US-00001 TABLE 1 Parental Strain strain Genotype Derivation or source S. enterica Wild type Wild type ATCC 14028 serovar Manassas, VA Typhimurium 14028 14028 zwf 14028 zwf Replacement of zwf YS1646 14028 msbB gene with zwf by (VNP20009) purI homologous YS1 14028 msbB1::tet recombination msbB1::tet Low et al., pp 47-59, In: Suicide Gene Therapy: Methods and Protocols, C. Springer (ed), Humana Press, 2003. Murray et al. 2001, J. Bacteriol. 183: 5554-5561. YS1 zwf YS1 zwf: Tn5 (Kan.sup.R) P22 zwf: Tn5 (Kan.sup.R)X msbB1::tet YS1 .fwdarw. Kan.sub.20.sup.r Murray somA YS873 14028 zbj10: Tn10 et al. 2001, J. msbB1::tet Bacteriol. 183: somA zbj10: Tn10 zwf: 5554-5561. Tn5 YS873 zwf YS873 (Kan.sup.R) P22 zwf: Tn5 (Kan.sup.R)X YS873 .fwdarw. Kan.sub.20.sup.r
[0178] CO.sub.2 resistant mutants were obtained as outlined in
[0179] The library was screened by plating dilutions of the library onto MSB agar and incubating them in 5% CO.sub.2 at 37 C. overnight. In particular, colonies were tested for CO.sub.2 resistance by plating serial dilutions of the library of bacteria onto MSB plates and incubating the plates at 37 C. in either ambient air or in air with 5% CO.sub.2. Colonies that were recovered from MSB plates incubated at 37 C. in air with 5% CO.sub.2 were deemed resistant to CO.sub.2. The resistance of these colonies to CO.sub.2 could be due to either the presence of the transposon insertion or to a spontaneous mutant. In order to eliminate any background of spontaneous mutants, the CO.sub.2-resistant colonies were pooled, P22 lysates prepared (P22 phage transduction by the method of Davis et al., 1980, Advanced Bacterial Genetics, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.), and the Tn5 insertions transferred to YS1646 and plates for individual colonies in MSB-kanamycin at 37 C. in ambient air. Individual colonies were then gridded and replica plated to MSB plates and incubated at 37 C. in either ambient air or in air with 5% CO.sub.2. Those colonies which tested positive by the replica plating were chosen for further study of retransduction to confirm the phenotype. These clones were also tested to ensure that there was no significant increase in TNF- induction using standard techniques such as those described by Low et al., 1999. The CO.sub.2 resistant clones chosen to undergo further testing include the clones designated 14.2, 32.2 and 37.2.
[0180] An example of the CO.sub.2 sensitivity and resistance observed using the plating efficiency method is shown in
[0181] Suppression of CO.sub.2-Mediated Growth Inhibition by zwf.sup. Mutants.
[0182] In order to further analyze the effect of the zwf mutation on growth of the msbB.sup. Salmonella, growth rates under different conditions were studied. To generate growth curves, 3 ml broth tubes were inoculated with single colonies and grown on a shaker overnight at 37 C. An adequate amount of LB or LB-0 broth was then inoculated 1:1000 with cells. Cells were held on ice until all inoculations were completed. Triplicate 3 ml aliquots were then placed in a 37 C. shaker with 250 rpm in air or 5% CO.sub.2. O.D..sub.600 was measured every 60 minutes and dilutions of bacteria were plated onto MSB or LB agar plates to calculate the number of colony forming units (CFU) per ml.
[0183]
[0184] Suppression of CO.sub.2 Mediated Cell Lysis by zwf.sup. Mutants.
[0185] To test if the decrease in CFU observed in YS873 in LB in the presence of 5% CO.sub.2 resulted from cell lysis, release of -galactosidase (a cytoplasmic enzyme not normally present in the culture supernatant) was determined. For -galactosidase expression, lacZ was cloned into the high copy vector pSP72 (Promega) and screened for bright blue colonies on LB agar containing 40 g/ml X-gal. -gal assays were performed according to the instructions for the Galacto-Star chemiluminescent reporter gene assay system (Applied Biosystems, Bedford, Mass.). Briefly, 1 ml of bacterial culture expressing -Gal from pSP72 was pelleted at 13,000g for 5 min. Supernatants were filtered through a 0.2 mm syringe filter and then assayed immediately or frozen at 80 C. until assayed with no further processing. Cell pellets were quickly freeze-thawed and suspended in 50 l or 200 ml B-Per bacterial cell lysis reagent (Pierce Chemical) containing 10 mg/ml lysozyme (Sigma). Bacteria were allowed to lyse for 10-20 min. at room temperature and then placed on ice. All reagents and samples were allowed to come to room temperature before use. Filtered supernatants and bacterial lysates were diluted as needed in Galacto-Star Lysis Solution or assayed directly. 3-gal standard curves were made by preparing recombinant -gal (Sigma, 600 units/mg) to 4.3 mg/ml stock concentration in 1PBS. The stock was diluted in Lysis Solution to prepare a standard curve of 100 ng/ml-0.05 ng/ml in doubling dilutions. 20 ml of standard or sample was added to each well of a 96-well tissue culture plate. 100 ml of Galacto-Star Substrate diluted 1:50 in Reaction Buffer Diluent was added to each well and the plate rotated gently to mix. The plate was incubated for 90 minutes at 25 C. in the dark and then read for 1 second/well in an L-Max plate luminometer (Molecular Devices). Sample light units/ml were compared to the standard curve and values converted to units -gal/ml. Percent release of -gal was determined by dividing units/ml supernatant by total units/ml (units/ml supernatant+units/ml pellet). All samples were assayed in triplicate. As shown in
[0186] Suppression of Acidic pH Mediated Growth Inhibition by zwf Mutants.
[0187] To test if increased or reduced pH would reduce sensitivity to CO.sub.2, LB media was buffered to pH 6.6 or 7.6 and cultures were grown in the presence or absence of 5% CO.sub.2. As shown in
[0188] Suppression of Acidic pH Mediated Cell Lysis by zwf Mutants.
[0189] Release of -galactosidase from the cytosol of the bacteria was used to test if the decrease in CFU observed in YS873 in LB at pH 6.6+/ the presence of 5% CO.sub.2 resulted from cell lysis (
[0190] Determination of Osmolarity and Gluconate Sensitivity Properties of zwf Mutants.
[0191] Phenotypes of strains were determined by replica plating (
[0192] As shown in the replica series of
[0193] Isolation and Identification of Genes Involved in Resistance to Acid pH.
[0194] The Tn5 insertion library described above is screened to identify mutants with resistance to acidity
[0195] Isolation and Identification of Genes Involved in Resistance to Osmolarity.
[0196] The Tn5 insertion library described above is screened to identify mutants with resistance to osmolarity
Example 2. Incorporation of Stress Resistance Genes for Use in Genetically Stabilized and Isolated Strains with Defined Flagellar Antigens and their Use in Protection Against Avian Influenza and Highly Pathogenic Derivatives
[0197] Construction of an Antibiotic-Sensitive Non-Polar Deletion in zwf.
[0198] A non-polar deletion in zwf was generated by constructing a pCVD442 vector capable of deleting the entire zwf coding region by homologous recombination with the Salmonella chromosome (Donnenberg and Kaper, 1991 Infection & Immunity 59:4310-4317; Low et al., 2003, Methods in Molecular Medicine; Suicide Gene Therapy, C. Springer (ed), Humana Press, pp 47-59, expressly incorporated in their entirety herein). Primers for PCR were designed that would generate one product immediately upstream of the 5 ATG start codon and a separate product immediately downstream of the 3 stop codon of the zwf coding region. The two separate products could then be ligated sequentially into the pCVD442 vector. The primers were:
TABLE-US-00002 zwf-5-reverse: (SEQIDNo.:1) 5-GTGTGAGCTCGTGGCTTCGCGCGCCAGCGGCGTTCC AGC-3 (withaddedSacI) and zwf-5 forward: (SEQIDNo.:2) 5-GTGTGCATGCGGGGGGCCATATAGGCCGGGGATTTA AATGTCATTCTCCTTAGTTAATCTCCTGG-3 (withaddedSphI); and zwf-3 reverse: (SEQIDNo.:3) 5-GTGTGCATGCGGGGTTAATTAAGGGGGCGGCCGCAT TTGCCACTCACTCTTAGGTGG-3 and 3-forward: (SEQIDNo.:4) 5-GTGTGTCGACCCTCGCGCAGCGGCGCATCCGGA TGC-3.
[0199] The primers also generate internal NotI, PacI, SphI, SfiI, and SwaI in order to facilitate cloning of DNA fragments, such as the influenza H5 and N1 antigens into the zwf for stable chromosomal integration without antibiotic resistance. This vector is referred to as pCVD442-zwf. Presence of the deletion, in Amp.sup.S Suc.sup.R colonies, was detected with PCR using the following primers:
TABLE-US-00003 zwf-FL-forward: (SEQIDNo.:5) 5-ATATTACTCCTGGCGACTGC-3 and zwf-FL-reverse: (SEQIDNo.:6) 5-CGACAATACGCTGTGTTACG-3.
[0200] Determination of Improved Penetration and Persistence in Gut Tissues.
[0201] Standard methods are utilized to determine increased penetration and persistence in gut tissues. In all cases, comparison of different dose levels is performed, comparing the parental, CO.sub.2, acidic pH, and/or osmolarity sensitive strain with the CO.sub.2, acidic pH and/or osmolarity resistant strain(s). 1) Total recovery from gut material. Mice are orally administered the parental strain and the resistant strains at different dose levels. At fixed times between days 1 and 21 (e.g., d. 1, 7, 14 & 21) the mice are euthanized (avoiding CO.sub.2 asphyxiation) and their gut collected by dissection. The gut is then homogenized and serial dilutions plated for Salmonella on Salmonella selective media such as SS agar, bismuth sulfite agar, or Hecktoen enteric agar. The number of Salmonella present for the parental and resistant strains at different times and dosing levels can then be compared to demonstrate improved penetration and persistence in the gut. 2) Determination of gut lining-associated Salmonella. Mice are orally administered the parental strain and the resistant strains at different dose levels. At fixed times between days 1 and 21 (e.g., d. 1, 7, 14 & 21) the mice are euthanized (avoiding CO.sub.2 asphyxiation) and their gut collected by dissection. The gut is then repeated flushed with a saline solution containing 100 ug/ml of gentamicin, an antibiotic that does not enter cells and will therefore not kill any bacteria that have penetrated the gut mucosal cells. The gut is then washed with saline to remove traces of gentamicin, homogenized and serial dilutions plated for Salmonella on Salmonella selective media such as SS agar, bismuth sulfite agar, or Hecktoen enteric agar. The number of Salmonella present for the parental and resistant strains at different times and dosing levels can then be compared in order to demonstrate improved gut penetration and persistence in the gut at lower doses.
[0202] Determining Immune Response to H5N1-Expressing Bacteria.
[0203] Live bacterial vaccines for H5N1 influenza prophylaxis or treatment described by Bermudes (WO/2008/03908) are engineered as described above to have an additional mutation in a stress-resistance gene such as zwf. Experimental determination of vaccine activity is known to those skilled in the arts. By way of non-limiting example, determination of an antibody response is demonstrated.
[0204] 1) Vertebrate animals including mice, birds, dogs, cats, horses, pigs or humans are selected for not having any known current or recent (within 1 year) influenza infection or vaccination. Said animals are pre-bled to determine background binding to, for example, H5 and N1 antigens.
[0205] 2) The Salmonella expressing H5 and N1 are cultured on LB agar overnight at 37. Bacteria expressing other H and or N antigens may also be used.
[0206] 3) The following day the bacteria are transferred to LB broth, adjusted in concentration to OD.sub.600=0.1 (210.sup.8 cfu/ml), and subjected to further growth at 37 on a rotator to OD.sub.600=2.0, and placed on ice, where the concentration corresponds to approx. 410.sup.9 cfu/ml.
[0207] 4) Following growth, centrifuged and resuspended in 1/10 the original volume in a pharmacologically suitable buffer such as PBS and they are diluted to a concentration of 10.sup.4 to 10.sup.9 c.f.u./ml in a pharmacologically suitable buffer on ice, warmed to room temperature and administered orally or intranasally in a volume appropriate for the size of the animal in question, for example 50 l for a mouse or 10 to 100 ml for a human. The actual dose measured in total cfu is determined by the safe dose as described elsewhere in this application.
[0208] 5) After 2 weeks, a blood sample is taken for comparison to the pretreatment sample. A booster dose may be given. The booster may be the same as the initial administration, a different species, a different serotype, or a different flagellar antigen (H1 or H2) or no flagellar antigen.
[0209] 6) After an additional 2 to 4 weeks, an additional blood sample may be taken for further comparison with the pretreatment and 2-week post treatment.
[0210] 7) A comparison of preimmune and post immune antibody response is performed by immunoblot or ELISA. A positive response is indicated by a relative numerical value 2 greater then background/preimmune assay.
[0211] Immunization with H5N1 Bacterial Vaccine Strains.
[0212] Live bacterial vaccines for H5N1 influenza prophylaxis or treatment described by Bermudes (WO/2008/03908) are engineered as described above to have an additional mutation in a stress-resistance gene such as zwf. An experiment to determine if H5N1 strains of Salmonella are capable of providing protection from challenge with the wildtype strain. Ducks are immunized orally with 510.sup.9 cfu of bacteria when 4 weeks old, then challenged with the standard challenge model of avian influenza at 6-weeks age.
[0213] Birds in Group A are immunized with empty vector. Group B receive Salmonella H5N1. Group C is immunized with Salmonella expressing the Tamiflu resistant neuraminidase mutations. Birds in Group D are not immunized. Each group is further divided into +/ Tamiflu treatment. Results of these experiments can be used to demonstrate the effectiveness of the vaccine on Tamiflu resistant strain, with and without Tamiflu treatment.
[0214] Immunization with a Trimeric Hemagglutinin Antigen.
[0215] The bacteria described above in Immunization with H5N1 Bacterial Vaccine Strains, are further engineered to contain a trimeric immunogen described by Wei et al. 2008 (J Virology 82: 6200-6208, expressly incorporated by reference in its entirety herein). The antigen is further modified to contain the HlyA C-terminal 60 amino acids in-frame, in order to guide secretion together with HlyBD (and a functional tolC). Immunization and efficacy evaluations are performed as described above.
[0216] Control of Bacterial Infection with Gluconate.
[0217] As described herein, in an msbB.sup. zwf.sup. strains are sensitive to physiological concentrations of CO.sub.2 in the presence of gluconate. The ability of gluconate to control excessive bacterial infections, such as might occur in a patient who becomes immunocompromised or otherwise has their health complicated such that the proliferation of the bacteria requires control, can be modeled using immunocompromised mice, such as nude (nu/nu) or severe combined immunodeficient (SCID) mice.
[0218] 1) The msbB.sup. zwf.sup. Salmonella cultured on LB agar overnight at 37. 2) The following day the bacteria are transferred to LB broth, adjusted in concentration to OD.sub.600=0.1 (210.sup.8 cfu/ml), and subjected to further growth at 37 on a rotator to OD.sub.600=2.0, and placed on ice, where the concentration corresponds to approx. 410.sup.9 cfu/ml.
[0219] 3) Following growth, centrifuged and resuspended in 1/10 the original volume in a pharmacologically suitable buffer such as PBS and they are diluted to a concentration of 10.sup.4 to 10.sup.9 c.f.u./ml in a pharmacologically suitable buffer on ice, warmed to room temperature and administered orally or intranasally in a volume appropriate for the size of the animal in question, for example, not more than 10 l/g body weight for a mouse. The actual dose measured in total cfu is determined by the safe dose as described by Bermudes (WO/2008/03908), depending upon the strain of bacteria.
[0220] 4) A dose response of 1, 5, 50 or 100 mg/mouse (50 mg, 250 mg, 2.5 g, 5 g/kg) of gluconate is given either orally or by intravenous administration.
[0221] 5) After 1 week, a blood or tissue sample is taken for comparison to the pretreatment sample using a colony forming unit assay.
[0222] 6) Survival of the mice is monitored over time.
OTHER EMBODIMENTS
[0223] Other embodiments are within the claims set forth below. For example, the host bacterium can be E. coli or any other lipid mutant or non-lipid mutant enteric bacterium found to be sensitive to CO.sub.2, acidic pH and/or osmolarity, including those of the genera Salmonella, Bordetella, Shigella, Yersenia, Citrobacter, Enterobacter, Klebsiella, Morganella, Proteus, Providencia, Serratia, Plesiomonas, and Aeromonas, all of which are known or believed to have cell wall structures similar to E. coli and Salmonella.
[0224] The various aspects of the disclosure may be combined and subcombined to represent all consistent combinations and subcombinations without departing from the scope of the invention. The invention is limited by neither the specific embodiments of the specification, nor the particular scope of the claims, but rather is to be treated as encompassing the full scope of each aspect disclosed, and the various combinations and permutations, which do not depart from the enabled disclosure herein.