Immunization and/or treatment of parasites and infectious agents by live bacteria

Abstract

Chimeric proteins are expressed, secreted or released by a bacterium to immunize against or treat a parasite, infectious disease or malignancy. The delivery vector may also be attenuated, non-pathogenic, low pathogenic, or a probiotic bacterium. The chimeric proteins include chimeras of, e.g., phage coat and/or colicin proteins, bacterial toxins and/or enzymes, autotransporter peptides, lytic peptides, multimerization domains, and/or membrane transducing (ferry) peptides. The active portion of the immunogenic chimeric proteins can include antigens against a wide range of parasites and infectious agents, cancers, Alzheimer's and Huntington's diseases, and have enhanced activity when secreted or released by the bacteria, and/or have direct anti-parasite or infectious agent activity. The activity of the secreted proteins is further increased by co-expression of a protease inhibitor that prevents degradation of the effector peptides. Addition of an antibody binding or antibody-degrading protein further prevents the premature elimination of the vector and enhances the immune response.

Claims

1. A genetically engineered chimeric peptide, comprising: a therapeutic peptide portion selected from the group consisting of: a Bacillus thuringiensis toxin, a Photorhabdus species insecticidal cytotoxin, a Xenorhabdus species insecticidal cytotoxin, anthelmintic cyclic heptapeptide segetalin D, and a cyclodepsipeptid, effective to provide a selective cytotoxic therapy of a parasitic disease caused by a parasite, the therapeutic peptide portion being toxic to the parasite; a targeting peptide portion comprising a parasite targeting (binding) peptide, effective to selectively target the therapeutic peptide portion to at least one cell of the parasite; and a secretion peptide portion selected from the group consisting of an autotransporter peptide, an HlyA peptide, an HlyB peptide, a type I secretion system active peptide, a type III secretion system active peptide, a colicin release peptide, a bacteriophage release peptide, Lpp-OmpA fusion, M13pIII, and a C-terminal RTC protein, effective to cause secretion of the chimeric peptide from a genetically engineered microorganism which produces the chimeric peptide, wherein the therapeutic peptide portion, the targeting peptide portion, and the secretion peptide portion of the chimeric peptide are derived by gene fusion from different genes.

2. The genetically engineered chimeric peptide according to claim 1, in combination with the genetically engineered microorganism, which is adapted for therapeutic administration to a mammal, wherein the chimeric peptide is produced based on at least one genetically engineered construct is selected from the group consisting of at least one of a plasmid and a chromosomal integration vector.

3. The genetically engineered chimeric peptide according to claim 2, wherein the secretion peptide portion is effective to cause secretion of the chimeric peptide from a genetically bacterial engineered microorganism.

4. The genetically engineered chimeric peptide according to claim 3, wherein the genetically engineered microorganism comprises an organism selected from the group consisting of: Lactobacillus sp., Lactococcus sp., Leuconostoc sp., Pediococcus sp., Streptococcus sp. Lactococcus sp., Bacillus sp., Bifidobacterium sp., Bacteroides sp., Escherichia coli., and Salmonella sp.

5. The genetically engineered chimeric peptide according to claim 2, wherein the genetically engineered construct comprises at least one ribosome binding site.

6. The genetically engineered chimeric peptide according to claim 1, wherein the chimeric peptide comprises a protease cleavage site between the targeting peptide portion and the secretion peptide portion.

7. The genetically engineered chimeric peptide according to claim 2, wherein the secretion peptide portion comprises a colicin peptide, and the at least one genetically engineered construct further encodes a colicin release protein, expressed in trans with respect to the chimeric peptide.

8. The genetically engineered chimeric peptide according to claim 1, produced by a genetically engineered microorganism, based on at least one genetically engineered construct which is polycistronic, comprising a plurality of genes separated by respective ribosomal binding sites.

9. The genetically engineered chimeric peptide according to claim 1, further comprising a protease inhibitor and at least two flanking protease cleavage sites.

10. The genetically engineered chimeric peptide according to claim 1, produced by a genetically engineered microorganism, adapted for therapeutic administration to a mammal, wherein the chimeric peptide is transcribed from at least one genetically engineered construct, which also encodes at least one protease inhibitor which is secreted from the genetically engineered microorganism within the mammal along with the chimeric peptide.

11. A chimeric peptide produced in situ in a mammal by a genetically engineered organism comprising at least one genetically engineered construct encoding the chimeric peptide, for treating a disease in the mammal caused by a parasite, by administration of the genetically engineered organism to the mammal, the chimeric peptide comprising: a therapeutic peptide portion selected from the group consisting of a Bacillus thuringiensis toxin, a Photorhabdus species insecticidal cytotoxin, a Xenorhabdus species insecticidal cytotoxin, an anthelmintic cyclic heptapeptide segetalin D, and a cyclodepsipeptid, effective to effect a selective cytotoxic therapy which is toxic to the parasite; a targeting peptide portion comprising a parasite targeting (binding) peptide effective to selectively target the therapeutic peptide portion to at least one cell of the parasite; and a secretion peptide portion selected from the group consisting of an autotransporter peptide, an HlyA peptide, an HlyB peptide, a type I secretion system active peptide, a type III secretion system active peptide, a colicin release peptide, a bacteriophage release peptide, Lpp-OmpA fusion, M13pIII, and a C-terminal RTC protein, effective to cause secretion of the chimeric peptide from the genetically engineered microorganism, wherein the therapeutic peptide portion, the targeting peptide portion, and the secretion peptide portion of the chimeric peptide are derived by gene fusion from different genes.

12. The chimeric peptide according to claim 11, in combination with the genetically engineered microorganism, comprising an organism selected from the group consisting of: Lactobacillus sp., Lactococcus sp., Leuconostoc sp., Pediococcus sp., Streptococcus sp. Lactococcus sp., Bacillus sp., Bifidobacterium sp., Bacteroides sp., Escherichia coli, and Salmonella sp.

13. The chimeric peptide according to claim 11, wherein the chimeric peptide comprises at least one protease cleavage site between the targeting peptide portion and the secretion peptide portion.

14. The chimeric peptide according to claim 11, wherein the at least one genetically engineered construct is polycistronic, comprising a plurality of genes separated by respective ribosomal binding sites, and the at least one genetically engineered construct further encodes a release protein, expressed in trans with respect to the chimeric peptide, adapted to facilitate a release of the chimeric peptide from the genetically engineered microorganism.

15. The chimeric peptide according to claim 11, wherein the at least one genetically engineered construct further encodes at least one secreted protease inhibitor.

16. A chimeric peptide, in a pharmaceutically acceptable form adapted to be administered orally, nasally, intravessically, via suppository, parenterally, intravenously, intramuscularly, intralymphaticly, intradermally, or subcutaneously, to a mammal as a treatment for a pathology caused by a parasite, resulting from translation of at least one genetically engineered heterologous construct encoding the chimeric peptide, having a plurality of distinct portions translated in-frame, comprising: a therapeutic peptide portion which is toxic to the parasite, selected from the group consisting of: a Bacillus thuringiensis toxin, a Photorhabdus species insecticidal cytotoxin, a Xenorhabdus species insecticidal cytotoxin, anthelmintic cyclic heptapeptide segetalin D, and a cyclodepsipeptid a targeting peptide portion comprising a parasite membrane binding peptide, effective to selectively target the therapeutic peptide portion to the parasite; and a secretion peptide portion selected from the group consisting of an autotransporter peptide, an HlyA peptide, an HlyB peptide, a type I secretion system active peptide, a type III secretion system active peptide, a colicin release peptide, a bacteriophage release peptide, Lpp-OmpA fusion, M13pIII, and a C-terminal RTC protein, effective to cause secretion of the chimeric peptide from the genetically engineered microorganism, wherein the therapeutic peptide portion, the targeting peptide portion, and the secretion peptide portion of the chimeric peptide are derived by gene fusion from different genes.

Description

5. BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows chimeric secreted protease inhibitors.

(2) FIG. 2 shows chimeric secreted antigens.

(3) FIG. 3 shows chimeric secreted lytic and therapeutic peptides.

6. DETAILED DESCRIPTION OF THE INVENTION

(4) The present invention provides, according to various embodiments, improved live attenuated therapeutic bacterial strains that express one or more therapeutic molecules together with one or more protease inhibitor polypeptides that inhibit local proteases that could deactivate the therapeutic molecules. In particular, one aspect of the invention relates to live attenuated bacterial strains that may include Salmonella vectoring novel chimeric antigens and/or anti-infective toxins to an individual to elicit a therapeutic response against an infectious disease. The types of infectious diseases may generally include prions, viruses, bacteria, protozoans (protists), fungi and helminthes (Mandell, Bennett and Dolin 2010, Principles and Practices of Infectious Diseases, 7.sup.th Edition, Elsiever Publishers, 4320 pages). Another aspect of the invention relates to reducing or eliminating the bacteria's ability to undergo conjugation, further limiting incoming and outgoing exchange of genetic material.

(5) For reasons of clarity, the detailed description is divided into the following subsections: protease sensitivity; protease inhibitors; antigens, lytic peptides, anti-infective proteins, targeting ligands, limiting conjugation and characteristics of some embodiments of the invention.

6.1. Protease Sensitivity

(6) The therapeutic proteins of some embodiments of the invention, including protease inhibitors, antigens, lytic peptides and therapeutic peptides, may be sensitive to proteases that exist at the site of infection, or from or within the infectious agent itself (e.g., Wanyiri et al., Infect Immun. 2007 January; 75(1): 184-192). Proteases may be classified by several different systems, for example, into six groups: serine proteases, threonine proteases, cysteine proteases, aspartate proteases, metalloproteases and glutamic acid proteases. Alternatively, proteases may be classified by the optimal pH in which they are active: acid proteases, neutral proteases, and basic proteases (or alkaline proteases). Well known proteases of the gut include trypsin, chymotrypsin, pepsin, carboxypeptidases and elastases. Other proteases such as furin, plasmin and lysosomal proteases and cathepsins may also be present. The protease sensitive proteins may also have protease cleavage sites that are artificially added to the protein being expressed. Assay of protease sensitivity is known to those skilled in the art.

6.2. Protease Inhibitors

(7) Protease inhibitors of some embodiments of the invention are preferably based on known or novel polypeptide inhibitors. The inhibitors include both synthetic peptides and naturally occurring, endogenous peptides. Classes of proteases are: cysteine protease inhibitors, serine protease inhibitors (serpins), trypsin inhibitors, Kunitz STI protease inhibitor, threonine protease inhibitors, aspartic protease inhibitors, metalloprotease inhibitors. Protease inhibitors can also be classified by mechanism of action as suicide inhibitors, transition state inhibitors, protein protease inhibitor (see serpins) and chelating agents. The protease inhibitors of some embodiments of the invention are protein or polypeptide inhibitors encoded by DNA contained within the bacteria.

(8) To result in the desired activity, the protease inhibitor peptides should be released or secreted outside of the bacteria, or displayed on the bacterial surface. Accordingly, the protease inhibitory peptides are modified by fusing them to secretion signals or co-expressed with colicin or bacteriophage lytic proteins as shown in FIG. 1. The secretion signals may be either N-terminal (derived from colicins, LPP:OmpA, M13pIII) or C-terminal (last 60 amino acids of an RTX protein such as the E. coli HlyA hemolysin, together with the required HlyBD supplied in trans and endogenous tolC). The secretion system may also be the autotransporter system, resulting in either surface displayed or released protease inhibitor. The N-terminal signal sequences are well known and characterized by the presence of a signal sequence cleavage site for an endogenous bacterial protease. Release may be further affected by co-expression of a colicin release protein. Thus, N-terminal signal sequences provide protease inhibitors, free from the signal sequence. The C-terminal signal sequence may be further engineered to have a protease cleavage site in between the protease inhibitory peptide and the signal sequence, such as a trypsin cleaveage signal (FQNALLVR, SEQ ID NO:47). The cleavage site may be for the same protease that the peptide inactivates. Thus, the protease activates its own inhibitor. The protease cleavage site may also be for a protease other than for the protease inhibitor, thus deactivating another protease. Multiple protease inhibitor peptides may be used in-frame with multiple protease cleavage signals (polymeric protease activated protease inhibitors), where the inhibitors alternate with cleavage sites. The polymeric protease activated protease inhibitors can be homo- or hetero-inhibitor polymers (i.e., have inhibitors for the same or different proteases, respectively), and/or homo- or hetero-protease cleavage polymers (i.e., have the same or different protease cleavage sites). Assay of protease inhibitors is known to those skilled in the art.

(9) Protease inhibitors have been reviewed by Laskowski and Kato, 1980, (Annual Review of Biochemistry 49: 593-626). Serine proteases inhibitors, the largest group, include 1) bovine pancreatic trypsin inhibitor (Kunitz) family, 2) pancreatic secretory trypsin inhibitor (Kazal) family, 3) Streptomyces subtilisin inhibitor family, 4) soybean trypsin inhibitor (Kunitz) family, 5) soybean proteinase inhibitor (Bowman-Birk) family 6) potato I inhibitor family, 7) potato II inhibitor family, 8) Ascaris trypsin inhibitor family, and 9) others. Protease inhibitors have also been grouped within the MEROPS peptidase database (Rawlings et al., 2008 Nucleic Acids Res. 36 Database issue, D320-325).

(10) Specific examples of protease inhibitors that may be expressed as complete proteins or peptide fragments corresponding to the active inhibitory site include but are not limited to aprotinin, cathepsin inhibitor peptide sc-3130, Niserria protease inhibitor, lymphocyte protease inhibitor, maspin, matrix metalloprotease inhibitors, macroglobulins, antithrombin, equistatin, Bowman-Birk inhbitor family, ovomucoid, ovoinhibitor-proteinase inhibitors from avian serum, dog submandibular inhibitors, inter-a-trypsin inhibitors from mammalian serum, chelonianin from turtle egg white, soybean trypsin inhibitor (Kunitz), secretory trypsin inhibitors (Kazal) a.sub.i-proteinase inhibitor, Streptomyces subtilisin inhibitor, plasminostreptin, plasmin inhibitor, factor Xa inhibitor, coelenterate protease inhibitors, protease inhibitor anticoagulants, ixolaris, human Serpins (SerpinA1(alpha 1-antitrypsin), SerpinA2, SerpinA3, SerpinA4, SerpinA5, SerpinA6, SerpinA7, SerpinA8, SerpinA9, SerpinA10, SerpinA11, SerpinA12, SerpinA13, SerpinB1, SerpinB2, SerpinB3, SerpinB4, SerpinB5, SerpinB6, SerpinB7, SerpinB8, SerpinC1 (antithrombin), SerpinD1, SerpinE1, SerpinE2, SerpinF1, SerpinF2, SerpinG1, SerpinN11, SerpinN12), cowpea trypsin inhibitor, onion trypsin inhibitor, alpha 1-antitrypsin, Ascaris trypsin and pepsin inhibitors, lipocalins, CI inhibitor, plasminogen-activator inhibitor, collegenase inhibitor, Acp62F from Drosophila, bombina trypsin inhibitor, bombyx subtilisin inhibitor, von Willebrand factor, leukocyte secretory protease inhibitor. Short peptide inhibitors of protease are preferred. Many protease inhibitors have one or more disulfide bonds. Fusion to thioredoxin (trxA) is known to improve protease inhibitor activity (e.g., Furuki et al., 2007, Fukuoka University Science Reports (Vol. 37, No. 1, Heisei 19 September) 37: 37-44). Fusion to glutathione-S transferase (GST) and co-expression with disulfide bond isomerase (DsbA) is also known to improve solubility. Examples of the peptide sequences of short peptide inhibitors are shown in Table 1.

(11) TABLE-US-00001 TABLE1 Sequencesofshortproteaseinhibitorpeptides Protease Protease(s) Protein/PeptideNameand/or Inhibitor inhibited PeptideSequence Leupeptin calpain, Leupeptin plasmin, trypsin, papain,and cathepsinB Aprotinin Trypsin RPDFCLEPPYTGPCKARIIRYFYNAKAGLCQTFVY Plasmin GGCRAKRNNFKSAEDCMRTCGGA Tissue SEQIDNO:1 kallikrein Aprotinin Variable Brinkmannetal,1991EurJ.Biochem202:95-99 homologues Protease Trypsin Syntheticpeptide:CFPGVTSNYLYWFK,SEQIDNO:48, Inhibitor15 correspondingtoaminoacids245-258ofhuman proteaseinhibitor. Tissue Serine DSLGREAKCYNELNGCTKIYDPVCGTDGNTYPNECVLCF protease protease ENRKRQTSILIQKSGPC inhibitor inhibitor, SEQIDNO:2 Kazaltype1, mature Furin Furin PAAATVTKKVAKSPKKAKAAKPKKAAKSAAKAVKPK inhibitors SEQIDNO:3 TKKVAKRPRAKRAA SEQIDNO:4 TKKVAKRPRAKRDL SEQIDNO:5 GKRPRAKRA SEQIDNO:6 CKRPRAKRDL SEQIDNO:7 CVAKRPRAKRDL SEQIDNO:8 CKKVAKRPRAKRDL SEQIDNO:9 RRRRRRL6R(hexa-L-arginine) SEQIDNO:10 Kallikrein Kallikrein2 SRFKVWWAAG Inhibitors SEQIDNO:11 AARRPFPAPS SEQIDNO:12 PARRPFPVTA SEQIDNO:13 Pepsinogen1-16 Pepsin LVKVPLVRKKSLRQNL SEQIDNO:14 Dunnetal.,1983BiochemJ209:355-362 Pepsinogen1-12 Pepsin LVKVPLVRKKSL SEQIDNO:15 Dunnetal.,1983BiochemJ209:355-362 Pepsinogen1-12 Pepsin LVKGGLVRKKSL(II)[Gly4,5] 4-7 SEQIDNO:16 substitution LVKVPGGRKKSL(III)[Gly6,7] SEQIDNO:17 LVKGGGGRKKSL(IV)[Gly4-7] SEQIDNO:18 Dunnetal.,1983BiochemJ209:355-362 Sunflowertrysin Trypsin GRCTKSIPPICFPD inhibitorSFTI-1 SEQIDNO:19 Odorranatrypsin Trypsin AVNIPFKVHFRCKAAFC inhibitor SEQIDNO:20 Ascaris Chymtrypsin GQESCGPNEVWTECTGCEMKCGPDENTPCP chymotrypsin Elastase LMCRRPSCECSPGRGMRRTNDGKOPASQCP elastaseinhibitor SEQIDNO:21 Ascaristrypsin Trypsin EAEKCBZZPGWTKGGCETCGCAQKIVPCTR inhibitor ETKPNPQCPRKQCCIASAGFVRDAQGNCIKFEDCPK SEQIDNO:22 Ascaristrypsin Trypsin EAEKCTKPNEQWTKCGGCEGTCAQKIVPCT inhibitor RECKPPRCECIASAGFVRDAQGNCIKFEDCPK SEQIDNO:23 Oniontrypsin Trypsin MKAALVIFLLIAMLGVLAAEAYPNLRQVVV inhibitor TGDEEEGGCCDSCGSCDRRAPDLARCECRD VVTSCGPGCKRCEEADLDLNPPRYVCKDMS FHSCQTRCSIL SEQIDNO:24 Barley Chymotrypsin MSSMEKKPEGVNIGAGDRQNQKTEWPELVGKSVEEAK chymotrypsin KVILQDKPAAQIIVLPVGTIVTMEYRIDRVRLFVDRL inhibitor2 DNIAQVPRVG SEQIDNO:25

6.3 Antigens

(12) Construction of chimeric bacterial proteins is used to adapt protein antigens such that they are released, surfaced displayed and/or secreted as shown in FIG. 2 to provide therapeutic molecules that are effective in eliciting an immune response. The antigens useful in some embodiments of the invention are known or novel proteins derived from infectious diseases (Mandell, Bennett and Dolin 2010, Principles and Practices of Infectious Diseases, 7.sup.th Edition, Elsevier Publishers, 4320 pages), or from cancer, Alzheimer's or Huntington's disease. Numerous specific antigens resulting in some degree of protective immunity have been described, e.g., WO/2009/150433 Flower et al., Antigenic Composition, expressly incorporated in its entirety herein. Epidermal growth factor receptors (EGFR) are known cancer antigens, beta-amyloid protein is a known antigen of Alzheimer's, and polyglutamine (polyQ) is a known antigen of Huntington's. However, there remains need to devise new ways improve upon the immune response. The antigens are secreted by bacteria using known secretion systems such as HlyA or autotransporters, or the novel colicin and M13 hybrids described herein. The antigens are expressed by the bacteria as described below from DNA constructs contained within the bacteria sufficient to result in the expression as described, and results in an improved immune response. Assay of antigenic responses are known to those skilled in the art, and is briefly described below.

6.4 Lytic Peptides

(13) As diagramed in FIG. 3, the antiparasitic proteins are expressed as fusions that are secreted, released or surface displayed. The activity of the antiparasitic proteins is improved by co-expression with one or more protease inhibitors. The desirability of combining protease inhibitors with lytic peptides has not previously been recognized as a means of both improving activity and specificity. Small lytic peptides (less than 50 amino acids) are used to construct chimeric proteins for more than one purpose. The chimeric proteins containing lytic peptides may be directly cytotoxic for parasites or infectious agents. In order to be cytotoxic they must be released, surface displayed and/or secreted (FIG. 3) and may be provided with cell specificity by the addition of a targeting ligand. Small lytic peptides have been proposed for use in the experimental treatment of parasites and infectious diseases. However, it is evident that most, if not all, of the commonly used small lytic peptides have strong antibacterial activity, and thus are not compatible with delivery by a bacterium (see Table 1 of Leschner and Hansel, 2004 Current Pharmaceutical Design 10: 2299-2310). Small lytic peptides useful in some embodiments of the invention are those derived from Staphylococcus aureus, S. epidermidis and related species, including the phenol-soluble modulin (PSM) peptides and delta-lysin (Wang et al., 2007 Nature Medicine 13: 1510-1514). The selection of the lytic peptide depends upon the primary purpose of the construct, which may be used in combination with other constructs providing other anticancer features. That is, the therapies provided in accordance with aspects of the present invention need not be provided in isolation, and the bacteria may be engineered to provide additional therapies or advantageous attributes. Constructs designed to be directly cytotoxic to cells employ the more cytoxic peptides, particularly PSM-alpha-3. Constructs which are designed to use the lytic peptide to affect escape from the endosome use the peptides with the lower level of cytotoxicity, such as PSM-alpha-1, PSM-alpha-2 or delta-lysin. Larger lytic peptides that may be used includes the actinoporins and equinatoxins from sea anemones or other coelenterates (Anderluh and Macek 2002, Toxicon 40: 111-124), are generally more potent than the bacterially-derived peptides, and are selected for use in being directly cytotoxic to parasites. Assay of lytic peptides is known to those skilled in the art.

(14) TABLE-US-00002 TABLE2 Membranelyticpeptidesusefulinsome embodimentsoftheinvention Peptideand source PeptideSequence Processed MAQDIISTISDLVKWIIDTVNKFTKK short active SEQIDNO:26 deltalysin Saureus Deltalysin MMAADIISTIGDLVKWIIDTVNKFKK processed SEQIDNO:27 Sepidermitidis Deltalysin MAQDIISTISDLVKWIIDTVNKFTKK fromCA-MRSA SEQIDNO:28 PSM-alpha-1 MGIIAGIIKVIKSLIEQFTGK SEQIDNO:29 PSM-alpha-2 MGIIAGIIKFIKGLIEKFTGK SEQIDNO:30 PSM-alpha-3 MEFVAKLFKFFKDLLGKFLGNN SEQIDNO:31 PSM-alpha-4 MAIVGTIIKIIKAIIDIFAK SEQIDNO:32 PSM-beta-1 MEGLFNAIKDTVTAAINNDGAKLGTSIVSIVEN GVGLLGKLFGF SEQIDNO:33 PSM-beta-2 MTGLAEAIANTVQAAQQHDSVKLGTSIVDIVAN GVGLLGKLFGF SEQIDNO:34 Actinoporins Lyticpeptidesfromseaanemones Equinatoxins andothercoelenterates

6.5 Anti-Infective Proteins

(15) As diagramed in FIG. 3, the antiparasitic proteins are expressed as fusions that are secreted, released or surface displayed. It has been known that certain bacteria such as Salmonella are capable of infecting certain roundworms, such as Caenorhabdities elegans (Lavigne et al., 2008, PLoS ONE 3:e3370; Gereven et al., 2007, FEMS Micobiol Lett 278: 236-241). However, it has not been suggested nor has it been recognized as desirable to construct an attenuated bacterium such as a Salmonella that could directly infect roundworms or other parasites following oral ingestion. Nor has it been suggested to engineer any such bacterium to directly attack roundworms or other parasites and to deliver therapeutic proteins that inhibit or kill the parasite. Nor would it have been understood that the activity of the antiparasitic proteins is improved by co-expression with one or more protease inhibitors. Furthermore, the fact that parasites in the process of infection, may cause the release of proteases that might deactivate the bacterially secreted proteins of some embodiments of the invention. Proteins with antiparasite activity include bacterial toxins with anti-insect and/or anti-parasite activity, including those from Bacillus thuringiensis (e.g., BT toxin) which have potential for treating parasites and infectious diseases (see Li et al., 2008, Biological Control, 47: 97-102; Li, et al., 2007, Plant Biotechnology Journal 5:455-464; Cappello, M. (2006) Proc. Natl. Acad. Sci. 103(41):15154-15159; Wei J. Z., 2003 Proc. Natl. Acad. Sci. 100:2760-2765, U.S. Pat. No. 5,651,965 Payne, Bacillus thuringiensis toxins and genes active against nematodes). Secreted insecticidal toxins and phenol oxidase inhibitors including but not limited to stilbenes from Photorhabdus and Xenorhabdus species are also encompassed by some embodiments of the invention. Lectins with antiparasite activity such those proteins purified from the corms of Pinellia ternata and Lycoris radiata. Both P. ternata agglutinin (PTA) protein and L. radiata agglutinin (LRA) as are also encompassed (Gaofu et al., 2008, Journal of Invertebrate Pathology 98: 40-45). Other proteins and peptides with anti-infective activity include the anthelmintic cyclic heptapeptide segetalin D (Dahiya 2007, Acta Pol. Pharm. 64: 509-516) cyclodepsipeptids (Dutton et al., J. Med. Chem. 46: 2057-2073) phenylalanine rich peptides, and toxins containing tyrosine and aspartic acid repeats (YD repeats).

6.6 Targeting Peptides

(16) As diagramed in FIG. 3, the anti-parasitic proteins are expressed as fusions that are secreted, released or surface displayed which may include targeting peptides. The activity of the anti-parasitic proteins with targeting peptides is improved by co-expression with one or more protease inhibitors. The targeting peptides are specific for the parasite to which the composition is directed. For example, phage display technology which is well known to those skilled in the art has been used to isolate peptides directed against Plasmodium, the causative agent of malaria (Lanzillotti et al., 2008, Trends In Parasitology 24: 18-23). Novel ligands may be isolated through standard phage display techniques (Barbass III et al., 2004, Phage Display, A Laboratory Manual, Cold Spring Harbor Press) including the use of commercially available kits (Ph.D-7 Phage Display Library Kit, New England Biolabs, Ipswich, Mass.). Thus for example, the peptide described by Li et al. (2008 Biochem Biophys Res Com 376: 489-493), ETTLKSF, SEQ ID NO:45, may be used as diagramed in FIG. 3 as an in-frame fusion for a bacterium directed toward malaria. Likewise, the peptide RGDS described by Quaissis et al. (1988 J. Protozool 35: 111-114) may be used as diagramed in FIG. 3 as an in-frame fusion for a bacterium directed toward leshmaniasis caused by Leishmania sp. Accordingly, known and novel peptides such as those determined through phage display to bind to a particular infectious agent are used in some embodiments of the invention.

6.7 Limiting Bacterial Conjugation

(17) The fertility inhibition complex (finO and finP), are cloned onto the chromosome using standard genetic techniques such that strains either with or without an F bacteria are not able to undergo bacterial conjugation. Other known inhibitory factors may also be used.

6.8 Characteristics of Therapeutic Bacteria Co-Expressing Protease Inhibitors with Chimeric Antigens, Lytic and Therapeutic Proteins

(18) The primary characteristic of the bacteria of certain embodiments of the invention is the enhanced effect of the effector molecule antigen, lytic peptide or anti-parasitic peptide relative to the parental strain of bacteria without expressing one or more protease inhibitors. In one embodiment, the percent increase in effect 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 parental strain of bacteria without expressing one or more protease inhibitors under the same conditions.

(19) A secondary characteristic of the bacteria of some embodiments of the invention is that they carry novel chimeric proteins that improve their function compared to other chimeric protein expression systems. In one embodiment, the percent improvement 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 another expression system under the same conditions.

(20) A third characteristic of the bacteria of some embodiments of the invention is that they carry novel chimeric proteins that prevent their elimination by antibodies compared to other chimeric protein expression systems. In one embodiment, the percent improvement 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 another expression system under the same conditions.

(21) Overall improvement is defined as an increase in effect, such as the ability to kill a parasite in vitro by the bacteria, or the amount of an antibody produced in vivo following administration with the bacteria expressing an antigen, with and without the protease inhibitor, and/or with and without an antibody inhibiting peptide. The effect of the protease inhibitor on protein therapeutic activity is determined using standard techniques and assays known to those skilled in the art. Inhibitors are expressed as secreted proteins as described above. Likewise, the effect of the antibody inhibitory protein on therapeutic activity is determined using standard techniques and assays known to those skilled in the art. Antibody inhibitors are expressed as native proteins (e.g., IgA protease in gram negative bacteria for vectors such as those using Salmonella, or spa, IdeS and EndoS in gram positive bacteria for vectors such as those using Streptococcus) or as secreted protein chimeras as described above. The contribution of the therapeutic protein, protease inhibitors and/or antibody inhibitory proteins is determined individually and in combination. Additivity, synergy or antagonism may determined using the median effect analysis (Chou and Talaly 1981 Eur. J. Biochem. 115: 207-216) or other standard methods.

7. FIGURE LEGENDS

(22) FIG. 1. Secreted protease inhibitors (PIs).

(23) A) A PI followed by the hlyA C-terminal signal sequence.

(24) B) A PI followed by an intervening protease cleavage site (downward arrow) and the hlyA C-terminal signal sequence.

(25) B) Multiple protease inhibitor peptides may be used in-frame with multiple protease cleavage signals (polymeric protease activated protease inhibitors), where the inhibitors alternate with cleavage sites. The polymeric protease activated protease inhibitors can be homo- or hetero-inhibitor polymers (i.e., have multiple inhibitors for the same or different proteases, respectively), and/or homo- or hetero-protease cleavage polymers (i.e., have multiple of the same or different protease cleavage sites). Thus, protease inhibitors 1, 2 and 3 can be the same protease inhibitor or different protease inhibitors, and the protease cleavage sites (downward arrows) can be the same protease cleavage side or different protease cleavage sites.

(26) C) A blocking peptide followed by an intervening protease cleavage site (downward arrow) and then the hlyA C-terminal signal sequence.

(27) D) The LPP:OmpA signal sequence followed by a protease inhibitor.

(28) E) The M13 pIII signal sequence (amino acids 1-18) followed by a protease inhibitor.

(29) F) An autotransporter cassette consisting of an autotransporter signal peptide, a protease inhibitor (passenger) followed by the autotransporter linker and (3-barrel.

(30) G) A pINIIIompA leader with a protease inhibitor (Longstaff et al., Biochemistry 1990 29: 7339-7347).

(31) H) A colicin N-terminal domain with a protease inhibitor.

(32) I) A thioredoxin (TrxA) fusion with a PI followed by the hlyA C-terminal signal sequence.

(33) J) A thioredoxin (TrxA) fusion with a PI followed by an intervening protease cleavage site (downward arrow) and the hlyA C-terminal signal sequence.

(34) K) A blocking peptide followed by a thioredoxin (TrxA) fusion with an intervening protease cleavage site (downward arrow) and then the hlyA C-terminal signal sequence.

(35) L) The LPP:OmpA signal sequence followed by a thioredoxin (TrxA) fusion with a protease inhibitor.

(36) M) The M13 pIII signal sequence followed by a thioredoxin (TrxA) fusion with a protease inhibitor.

(37) N) An autotransporter cassette consisting of an autotransporter signal peptide, a thioredoxin (TrxA) fusion and a protease inhibitor (passengers) followed by the autotransporter linker and -barrel.

(38) O) A pINIIIompA leader with a thioredoxin (TrxA) fusion with a protease inhibitor.

(39) P) A colicin N-terminal domain with a thioredoxin (TrxA) fusion with a protease inhibitor. Q) F) A colicin lysis protein that may be co-expressed in trans with any of the above.

(40) FIG. 2. Chimeric antigens.

(41) A) A colicin N-terminal domain fused in-frame with thioredoxin (TrxA) and an antigenic domain.

(42) B) An M13 pIII signal sequence with amino acids 1 to 18 followed by an antigen and then a membrane anchor truncated M13 pIII amino acids 19 to 372.

(43) C) An M13 pIII signal sequence with a membrane anchor truncated M13 pIII amino acids 1 to 372 and an antigen.

(44) D) An autotransporter cassette consisting of an autotransporter signal peptide, an antigen (passenger) followed by the autotransporter linker and (3-barrel.

(45) E) An antigen fused to the 60 C-terminal amino acids of HlyA (together with HlyBD and tolC in trans).

(46) F) A colicin lysis protein that may be co-expressed in trans with any of the above.

(47) FIG. 3. Lytic and therapeutic peptide chimeras.

(48) A) A lytic peptide followed by the hlyA signal sequence.

(49) B) A lytic peptide, parasite targeting (binding) peptide followed by an intervening protease cleavage site (downward arrow), hlyA signal peptide chimera.

(50) C) The M13 pIII signal sequence followed by a lytic peptide and the membrane anchor truncated M13 pIII amino acids 19 to 372.

(51) D) The M13 pIII signal sequence followed by a lytic peptide and the membrane anchor truncated M13 pIII amino acids 19 to 372 and a targeting peptide.

(52) E) The M13 PIII signal sequence followed by a targeting peptide, a lytic peptide and the membrane anchor truncated M13 pIII amino acids 19-372.

(53) F) The M13 pIII signal sequence followed by a lytic peptide.

(54) G) The M13 pIII signal sequence followed by a lytic peptide.

(55) H) A therapeutic peptide (e.g., BT toxin) fused to hlyA C-terminus.

(56) I) The M13 pIII signal sequence followed by a therapeutic peptide and the membrane anchor truncated M13 pIII amino acids 19 to 372.

(57) J) A colicin N-terminal domain followed by a therapeutic peptide.

(58) K) An autotransporter cassette consisting of an autotransporter signal peptide, a therapeutic peptide (passenger) followed by the autotransporter linker and (3-barrel.

(59) L) A colicin lysis protein that may be co-expressed in trans with any of the above.

8. EXAMPLES

(60) In order to more fully illustrate the invention, the following examples are provided.

8.1. Example 1: Methods for Obtaining Bacterial Strains with Suitable Genetic Backgrounds

(61) A first step in selection of an appropriate strain based upon the known species specificity (e.g, S. typhi is human specific and S. typhimurium has broad species specificity including humans, birds, pigs and many other vertebrates). Thus, if the target species for immunization were limited to humans, S. typhi would be appropriate. If more species are desired to be immunized including humans, birds, pigs, cattle, dogs, horses and many other vertebrates, then other serotypes may be used. For example, S. typhimurium and S. montevideo which have non-overlapping O-antigen presentation (e.g., S. typhimurium is O-1, 4, 5, 12 and S. typhi is Vi, S. montevideo is O-6, 7) are representative examples. Methods to genetically alter the serotype within a single strain are known to those skilled in the art, including Favre et al., 1997 WO 97/14782 Methods for delivering heterologous O-antigens; and Roland, 2000, WO/2000/004919). Thus, S. typhimurium is a suitable serotype for a prime/boost strategy where S. typhimurium is either the primary vaccine, or the booster vaccine where the primary vaccine is another serotype such as S. typhi or S. montevideo. Furthermore, both S. typhimurium and S. montevideo are suitable for humans, pigs, cattle or birds. A second step follows serotype selection where the first genetic mutation is introduced which may involve the use of antibiotic resistance markers and where any antibiotic resistance makers are then eliminated, followed by a third step where a second genetic mutation is introduced which may involve the use of antibiotic resistance markers and where any antibiotic resistance makers are then also eliminated. Reiteration of genetic deletion and antibiotic marker elimination can be used to supply additional mutations. Methods for reiterative chromosomal deletion and elimination of antibiotic resistance markers are known to those skilled in the art, including TN10 transposon deletion followed by Bochner selection (Bochner et al., 1980, J Bacteriol. 143: 926-933) for elimination of the tetracycline antibiotic resistance marker, lamda red recombinase deletion followed by flip recombinase elimination of the antibiotic resistance marker (Lesic and Rahme, 2008, BMC Molecular Biology 9:20), and suicide vectors such as those containing sucrase gene (e.g., pCVD442, Donnenberg and Kaper, 1991 Infect Immun 59: 4310-4317). Spontaneous mutations may also be rapidly and accurately selected for, such as the Suwwan, a large IS200-mediated deletion (Murray et al., 2004, Journal of Bacteriology, 186: 8516-8523). Thus, the starting strain can be a wild type Salmonella such as ATCC 14028, and the Suwwan, IS200 deletion selected for using chlorate (Murray et al., 2004, Journal of Bacteriology, 186: 8516-8523). A second mutation in msbB can be introduced using pCVD442 as described by Low et al., 2004, Methods Mol Med. 2004; 90:47-60). A third mutation can be generated in zwf as described by Karsten et al., 2009, BMC Microbiol. BMC Microbiol. 2009 Aug. 18; 9:170. Thus, the strain generated has deletions in the Suwwan region, msbB and zwf. In S. montevideo, where the Suwwan mutation is not known to occur, a pCVD442 vector is used to generate the equivalent mutation, together with the same procedures above (altered as necessary for DNA sequence variations in the DNA portions used for homologous recombination), resulting in a pair of strains having the same mutational background together with different bacterial antigens. These strains, alone or used for alternating doses, form a basic platform into which the antigens and protease inhibitor gene constructs are inserted.

8.2 Example 2: Production of Antigen Chimeras

(62) Chimeric antigens are generated using standard molecular genetic techniques, including synthetic biology (e.g., chemically synthesized oligonucleotides annealed into larger constructs forming entire genes based on the nucleic acid and/or amino acid sequence selected) and expressed in bacteria using methods known to those skilled in the art, operably linking a promoter, ribosomal binding site and initiating methionine if not provided by the first portion of the construct. The construct may either be an exogenous plasmid or a chromosomal or virulence (VIR) plasmid integration vector, for which many different integration sites exist, including but not limited to any of the attenuation mutations or any defective (incomplete) phage elements, intergenic regions or the IS200 elements. The constructs may also be polycistronic, having multiple genes and/or gene products separated by ribosomal binding sites. The downstream region may contain a termination signal (terminator). Antigen fusions are performed in-frame. Any infectious disease for which an antigenic determinant is known may be used, as exemplified in FIG. 2A. For example, if a vaccine for influenza is needed, A colicin N-terminal domain, such as amino acids 1-232 of colE3: (MSGGDGRGHNTGAHSTSGNINGGPTGLGVGGGASDGSGWSSENNPWGGGSGSGIHW GGGSGHGNGGGNGNSGGGSGTGGNLSAVAAPVAFGFPALSTPGAGGLAVSISAGALSA AIADIMAALKGPFKFGLWGVALYGVLPSQIAKDDPNMMSKIVTSLPADDITESPVSSLPL DKATVNVNVRVVDDVKDERQNISVVSGVPMSVPVVDAKPTERPGVFTASIPGAPVLNI) SEQ ID NO:35, is synthesized in frame with an antigen, such as the hemagglutinin from the H1N1 swine flu. The protein sequence for a portion of the swine flu hemagglutin, the HA1 fragment containing an initiating methionine and artificial second codon but without the initial signal sequence, an altered protease cleavage site and membrane anchor truncation (e.g., general antigen organization as described by Wei et al., 2008 J. Virology 82: 6200-6208) is given by the amino acid sequence:

(63) TABLE-US-00003 SEQIDNO:36 MATFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKL CKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETSSSDNGTCY PGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGA KSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSL YQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKIT FEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLP FQNIHPITIGKCPKYVKSTKLRLATGLRNVPSIQSTGLFGAIAGFIEGGW TGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFT AVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSN VKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSE EAKLNREEIDG

(64) A colicin release protein, such as that of colE3 (MKKITGIILLLLAVIILSACQANYIRDVQGGTVSPSSTAEVTGLATQ, SEQ ID NO:37) is expressed in trans in order to enhance secretion and/or release. Each of the genes may be localized to an exogenously introduced plasmid, the endogenous virulence (VIR) plasmid, or the chromosome, together as a polycistronic construct or separately as monocistronic constructs, within any of the deleted attenuating genes, IS200s, or intervening sequences as described for the functional insertion of the cytosine deaminase gene with an msbB deletion (King et al., 2009 Methods Mol Biol. 542: 649-59; Nemunaitis et al., 2003, Cancer Gene Therapy 10: 737-744). Bacteria expressing any of these constructs are tested for secretion into the media by the ability of an antibody to the bona fide antigen to react with the proteins of the supernatant using a standard immunological assay such as an immunoblot or enzyme linked immunosorbent assay (ELISA).

8.3 Example 3: Selecting Protease Inhibitors

(65) Protease inhibitors are generated using knowledge of the predicted proteolytic cleavage of the antigen or other effector molecule. For example, the ExPASy PeptideCutter tool: Gasteiger et al. (Protein Identification and Analysis Tools on the ExPASy Server, In: John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press, 2005) may be used to test the predicted proteolytic sensitivity of the antigen or other effector molecule. Using ExPASy, the hemagglutinin in the example above would be cleaved extensively by chymotrypsin (between 46 to 98 times depending on high specificity FYW not before P (46 times) or low specificity FYWML (SEQ ID NO:50) not before P (98 times), while there are no Factor Xa sites. Thus, since cleavage of the effector molecule has the potential to occur, chymotrypsin represent a protease target for which inhibition would improve the antigenicity or activity of a co-expressed molecule by inhibiting its destruction by proteolytic degradation, whereas Factor Xa is identified as a cleavage site that is not present, does not need to be inhibited, and whose cleavage recognition site could be added between protein domains where removal of a domain by proteolysis is desirable.

8.4 Example 4: Secreted Protease Inhibitors

(66) Secreted protease inhibitors are generated using standard molecular genetic techniques and expressed in bacteria using methods known to those skilled in the art, operably linking a promoter, ribosomal binding site and initiating methionine if not provided by the first portion of the construct. The construct may either be a plasmid or a chromosomal integration vector, for which many different integration sites exist, including but not limited to any of the attenuation mutations or any of the IS200 elements. The constructs may also be polycistronic, having multiple genes and/or gene products separated by ribosomal binding sites. The downstream region may contain a termination signal (terminator). Different forms of the protease inhibitor constructs are shown in FIG. 1. The constructs used have multiple forms, such as: FIG. 1A) a protease inhibitor such as the Ascaris chymotrypsin and elastase inhibitor GQESCGPNEV WTECTGCEMK CGPDENTPCP LMCRRPSCEC SPGRGMRRTN DGKCIPASQCP SEQ ID NO:38 followed by the C-terminal signal sequence of hlyA STYGSQDYLNPLINEISKIISAAGNLDVKEERSAASLLQLSGNASDFSYGRNSITLTASA, SEQ ID NO:39 or

(67) FIG. 1B) a protease inhibitor such as the Ascaris chymotrypsin and elastase inhibitor GQESCGPNEV WTECTGCEMK CGPDENTPCP LMCRRPSCEC SPGRGMRRTN DGKCIPASQCP, SEQ ID NO:40 followed by a factor Xa cleavage site (IEGR, SEQ ID NO:49) followed by the C-terminal signal sequence of hlyA STYGSQDYLNPLINEISKIISAAGNLDVKEERSAASLLQLSGNASDFSYGRNSITLTASA, SEQ ID NO:41, or

(68) FIG. 1E) An N-terminal signal sequence, such as that from M13pIII (MKKLLFAIPLVVPFYSHS SEQ ID NO:42), followed by a protease inhibitor such as the Ascaris chymotrypsin and elastase inhibitor GQESCGPNEV WTECTGCEMK CGPDENTPCP LMCRRPSCEC SPGRGMRRTN DGKCIPASQCP, SEQ ID NO:43.

(69) Several other secreted protease inhibitor forms are diagramed in FIG. 1, including the use of an autotransporter system and fusion with thioredoxin (trxA). A colicin release protein, such as that of colE3 (MKKITGIILLLLAVIILSACQANYIRDVQGGTVSPSSTAEVTGLATQ, SEQ ID NO:44) may be expressed in trans in order to enhance secretion and/or release. Bacteria expressing any of these constructs are tested for secretion into the media and the ability of the media to inhibit a protease such as chymotrypsin in a standard protease assay known to those skilled in the art. Many protease assays are commercially available, such as the QuantiCleave Fluorescent Protease Assay Kit, and QuantiCleave Protease Assay Kit II (Thermo/Fisher, Rockford, Ill.), Protease Assay Kit (G Biosciences, Maryland Heights, Mo.), PepTag Protease Assay (Promega, Madison, Wis.; 1993 Promega Notes Magazine 44: 2), Viral Protease Assay Kits (AnaSpec, Fremont, Calif.), Protease Assay Kit from Calbiochem (Calbiochem, San Diego, Calif.). Standard laboratory techniques to measure protease activity, and thus the reduced activity of protease inhibitors, include densitometric, spectrophotometric, colorometric and fluorometric assays, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE), two dimentional SDS-PAGE, high pressure liquid chromatography (HPLC) and mass spectroscopy (mass-spec).

8.5 Example 5: Determining Immune Response to an Influenza Hemagglutinin-Expressing Bacteria

(70) Experimental determination of vaccine activity is known to those skilled in the art. By way of non-limiting example, determination of an antibody response is demonstrated. It is understood that the resulting bacteria are then determined for LD.sub.50 using standard methods (e.g., Welkos and O'Brien, 1994, Determination of median lethal and infectious doses in animal model systems, Meth. Enzymol. 235:29-39) in order that the experiments proceed using safe doses. Translation to human studies is performed using multiples species (e.g., dogs, monkeys, pigs) and that a safe does is chosen well below the safe does in other species on either a mg/kg or mg/meter square.

(71) 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, hemagglutinin antigens.

(72) 2) The Salmonella expressing hemagglutinin are cultured on LB agar overnight at 37. Bacteria expressing the antigens in combination with a protease inhibitor may also be used.

(73) 3) The following day the bacteria are transferred to LB broth, adjusted in concentration to OD.sub.600=0.1 (210.sup.8 c.f.u./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 c.f.u./ml.

(74) 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 parenterally 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 by oral administration. The actual dose measured in total c.f.u. is determined by the safe dose as described above.

(75) 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 serotype and containing the same antigens (and/or protease inhibitors) as the initial administration, a different species, a different serotype, or a different flagellar antigen (H1 or H2) or no flagellar antigen.

(76) 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.

(77) 7) A comparison of preimmune and post immune antibody response is preformed by immunoblot or ELISA. A positive response is indicated 1) by a relative numerical value 20% or greater than background/preimmune assay with the antigen alone, and/or 2) by a relative numerical value 20% or greater than without the protease inhibitor.

8.6 Example 6: Immunization with a Hemagglutinin-Expressing Bacterial Vaccine Strains

(78) An experiment to determine if hemagglutinin-expressing strains of Salmonella are capable of providing protection from challenge with the wildtype strain with improvement from co-expression with protease inhibitors. Ducks are immunized orally with a tolerated dose of bacteria when 4 weeks old, then challenged with the standard challenge model of influenza at 6 weeks age.

(79) Birds in Group A are immunized with empty vector. Group B receive Salmonella expressing hemagglutinin. Group C is immunized with Salmonella expressing the protease inhibitor with no antigen. Group D is immunized with Salmonella expressing the hemagglutinin antigen and the protease inhibitor. Birds in Group E are not immunized. Comparative results of these experiments can be used to demonstrate the effectiveness of the vaccine with and without protease inhibitor.

8.7 Example 7: Therapeutic Peptides with Lytic Anti-Parasite Activity

(80) Therapeutic peptides are generated using standard molecular genetic techniques and expressed in bacteria using methods known to those skilled in the art, operably linking a promoter, ribosomal binding site and initiating methionine if not provided by the first portion of the construct. The construct may either be a plasmid or a chromosomal integration vector, for which many different integration sites exist, including but not limited to any of the attenuation mutations or any of the IS200 elements. The constructs may also be polycistronic, having multiple genes and/or gene products separated by ribosomal binding sites. The downstream region may contain a termination signal (terminator). Antigen fusions are performed in-frame.

(81) An example of an antigen fusion is given in FIG. 3B. The lytic peptide PSM-alpha-3 MEFVAKLFKFFKDLLGKFLGNN, SEQ ID NO:31 is fused to the malaria targeting peptide ETTLKSF, SEQ ID NO:45, followed by a factor Xa cleavage site (IEGR, SEQ ID NO:49) C-terminal signal sequence of hlyA STYGSQDYLNPLINEISKIISAAGNLDVKEERSAASLLQLSGNASDFSYGRNSITLTASA SEQ ID NO:46. A colicin release protein, such as that of colE3 may be expressed in trans in order to enhance secretion and/or release. Bacteria expressing any of these constructs are tested for secretion into the media by the ability of the media to kill a parasite, such as Plasmodium sp., the causative agents of malaria.

(82) It will be understood that the foregoing is only illustrative of the principles of the invention, and that various modifications can be made by those skilled in the art without departing from the scope and spirit of the invention.