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WO2022271976A1 - Salmonella atténuée, vaccins et plateforme d'administration - Google Patents

Salmonella atténuée, vaccins et plateforme d'administration Download PDF

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Publication number
WO2022271976A1
WO2022271976A1 PCT/US2022/034769 US2022034769W WO2022271976A1 WO 2022271976 A1 WO2022271976 A1 WO 2022271976A1 US 2022034769 W US2022034769 W US 2022034769W WO 2022271976 A1 WO2022271976 A1 WO 2022271976A1
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Prior art keywords
mutation
gene
salmonella
cya
attenuated salmonella
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PCT/US2022/034769
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English (en)
Inventor
Bijit Kumar BHOWMIK
Dharanesh Mahimapura GANGAIAH
Arvind Kumar
Jonathan Earl SNYDER
Original Assignee
Elanco Us Inc.
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Application filed by Elanco Us Inc. filed Critical Elanco Us Inc.
Publication of WO2022271976A1 publication Critical patent/WO2022271976A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A61K39/012—Coccidia antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/255—Salmonella (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1282—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Clostridium (G)
    • C—CHEMISTRY; METALLURGY
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/36—Adaptation or attenuation of cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/36011—Togaviridae
    • C12N2770/36041—Use of virus, viral particle or viral elements as a vector
    • C12N2770/36043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2820/00—Vectors comprising a special origin of replication system
    • C12N2820/60—Vectors comprising a special origin of replication system from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/42—Salmonella
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the therapeutic biomolecule can have desired effect such as treating or preventing an infection, disease or disorder, or eliciting protective immunity against a pathogenic or parasitic organism.
  • Salmonella is genus of Gram-negative facultative rod-shaped bacteria, which is the causative agent of salmonellosis in animals, birds, as well as in humans.
  • Two species of Salmonella include Salmonella enterica and Salmonella bongori.
  • a majority of infections are caused by Salomnella enterica serovar Typhimurium. These highly adaptive bacteria can survive and colonize in the hostile environment of the gastrointestinal tract.
  • Salmonella species are facultative intracellular pathogens and can invade different cell types, including epithelial cells, M cells, macrophages, and dendritic cells. .
  • Salmonella has evolved to withstand the attack by macrophages by colonizing inside them.
  • the genome of S. Typhimurium and its several isolates has been sequenced and is known, thus manipulation of the S. Typhiurium genome via genetic techniques and recombinant technology is feasible and has led to the generation and development of altered Salmonella for various uses and applications.
  • Attenuated Salmonella has been utilized in various approaches as a vaccine antigen delivery vehicle, with varied results (Roland KL and Brenneman KE (2013) Exp Rev Vaccines 12(9):1033- 1045; Clark-Curtiss JE and Curtiss R. (2016) J Immunol 200:39-48).
  • the heterologous gene is usually transformed as a multi-copy plasmid into the bacterial cell, which can be unstable.
  • An alternative approach is to integrate the relevant gene in the bacterial chromosome, which requires multiples steps and can result in various limitations.
  • an intracellular delivery platform wherein a genetically modified bacterium is utilized to deliver a therapeutic biomolecule, particularly a molecule having preventative or therapeutic anti-infective activity, one or more immunomodulatory factor, or one or more growth- promoting biomolecule.
  • the therapeutic biomolecule is delivered intracellularly.
  • the therapeutic biomolecule is delivered systemically to an animal.
  • the therapeutic biomolecule is delivered directly to the mucosa of an animal in need thereof.
  • the genetically modified bacterium is an attenuated bacterium.
  • the genetically-modified bacterium is an auxotrophic bacterium.
  • the bacterium may be a Salmonella bacterium, for example, an attenuated Salmonella bacterium.
  • the bacterium may be an attenuated Salmonella bacterium further genetically modified or mutated to have additional mutations beyond the attenuating mutations.
  • a live intracellular delivery platform comprising an attenuated Salmonella bacterium comprising nucleic acid encoding and/or expressing a therapeutic biomolecule, wherein the attenuated Salmonella bacterium comprises a mutation in at least one gene selected from cya, crp, phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, and aroA.
  • the attenuated Salmonella bacterium is a Salmonella enterica.
  • the attenuated Salmonella bacterium is a Salmonella enterica serovar Typhimurium strain.
  • the attenuated Salmonella bacterium comprises a mutation in at least one gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, and sspH2.
  • the attenuated Salmonella bacterium further comprises a mutation in at least one of a cya and crp gene.
  • the attenuated Salmonella bacterium further comprises a mutation in a cya gene and a crp gene.
  • the attenuated Salmonella bacterium comprises a mutation in at least one of cya and crp gene and further comprises a mutation in at least one gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, and sspH2.
  • the attenuated Salmonella bacterium comprises at least one first mutation and at least one second mutation; wherein the at least one first mutation is selected from a mutation in a cya and a crp gene; and the at least one second mutation is selected from a mutation in at least one gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, asd, and aroA.
  • the attenuated Salmonella bacterium comprises at least one first mutation and at least one second mutation; wherein the at least one first mutation is a mutation in cya and crp; and the at least one second mutation is selected from a mutation in at least one gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, asd, and aroA.
  • the attenuated Salmonella bacterium comprises at least one first mutation and at least one second mutation; wherein the at least one first mutation is a mutation in cya and crp; and the at least one second mutation is selected from a mutation in at least one gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sopF and feoABC.
  • the therapeutic biomolecule is a heterologous therapeutic biomolecule and is not natively encoded or expressed by the attenuated Salmonella bacterium.
  • the nucleic acid encoding and/or expressing a therapeutic molecule is self-amplifying nucleic acid.
  • the nucleic acid encoding and/or expressing a therapeutic molecule is provided on a plasmid or vector in the attenuated bacterium.
  • the nucleic acid encoding and/or expressing a therapeutic molecule is integrated in the genome or the attenuated bacterium.
  • a live intracellular delivery platform comprising an attenuated Salmonella bacterium comprising a self-amplifying nucleic acid is provided.
  • the attenuated Salmonella bacterium is a Salmonella enterica. In some embodiments, the attenuated Salmonella bacterium is a Salmonella enterica serovar Typhimurium strain.
  • the self-amplifying nucleic acid encodes for at least one alphavirus genome replication protein selected from nsP1, nsP2, nsP3, and nsP4. [00018] In some embodiments, the nucleic acid encodes a biomolecule having a therapeutic effect selected from at least one of an antibody, a ribonucleic acid (RNA), an anti-infective peptide, and an antigen. [00019] In some embodiments, the biomolecule having a therapeutic effect is a camelid antibody.
  • the camelid antibody is directed against a toxin of Clostridium perfringens.
  • the camelid antibody comprises SEQ ID NO: 169.
  • the biomolecule having a therapeutic effect is a small hairpin RNA (shRNA) or a short interfering RNA (siRNA).
  • the shRNA or siRNA comprises one or more of SEQ ID NO: 135-146.
  • the biomolecule having a therapeutic effect is an antigen derived from an Eimeria parasite.
  • the antigen is selected from at least one of Eimeria tenella elongation factor -1 ⁇ ; EtAMA1; Eimeria tenella 5401; Eimeria acervulina lactate dehydrogenase antigen gene; Eimeria maxima surface antigen gene; Glyceraldehyde 3-phosphate Dehydrogenase (GAPDH); Eimeria common antigen 14-3-3 wherein the antigen comprises one or more of SEQ ID NOs: 153-157 or 173-177.
  • the live intracellular delivery platform comprises an Eimeria antigen protein selected from EtAMA1, Glyceraldehyde 3-phosphate Dehydrogenase (GAPDH) and Eimeria common antigen 14-3-3, or an antigenic fragment or portion thereof.
  • Eimeria antigen protein selected from EtAMA1, Glyceraldehyde 3-phosphate Dehydrogenase (GAPDH) and Eimeria common antigen 14-3-3, or an antigenic fragment or portion thereof.
  • a live intracellular delivery platform wherein the attenuated Salmonella bacterium comprises a mutation in at least one gene selected from cya, crp, phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, and aroA.
  • a live intracellular delivery platform wherein the attenuated Salmonella bacterium comprises a mutation in at least one gene selected from cya, crp, phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, aroA, and thyA.
  • a live intracellular delivery platform wherein the attenuated Salmonella bacterium comprises a mutation in at least one gene selected from cya, crp, phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, and SspH2.
  • the attenuated Salmonella bacterium comprises a mutation further comprises a mutation in the asd gene.
  • the attenuated Salmonella bacterium comprises a mutation further comprises a mutation in at least one gene selected from paba, pabb, asd, and aroA.
  • a live intracellular delivery platform wherein the attenuated Salmonella bacterium comprises a mutation in cya and crp and further comprises a mutation in at least one gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, and SspH2.
  • a live intracellular delivery platform wherein the attenuated Salmonella bacterium comprises a mutation in cya and crp and further comprises a mutation in at least one gene selected from phoPQ, ompR-envZ, sifA, sopF, ompR- envZ, feoABC, and SspH2.
  • a live intracellular delivery platform is provided wherein the attenuated Salmonella bacterium comprises a mutation in cya and crp and further comprises a mutation in at least one gene selected from phoPQ, ompR-envZ, sifA, sopF, ompR-envZ and feoABC.
  • a live intracellular delivery platform wherein the attenuated Salmonella bacterium comprises a mutation in cya and crp and further comprises a mutation in at least one gene selected from phoPQ, ompR-envZ, sifA and sopF.
  • a live intracellular delivery platform is provided wherein the attenuated Salmonella bacterium comprises a mutation in cya and crp and further comprises a mutation in phoPQ.
  • a live intracellular delivery platform is provided wherein the attenuated Salmonella bacterium comprises a mutation in cya and crp and further comprises a mutation in sifA.
  • a live intracellular delivery platform wherein the attenuated Salmonella bacterium comprises a mutation in cya and crp and further comprises a mutation in ompR-envZ. In some embodiments, a live intracellular delivery platform is provided wherein the attenuated Salmonella bacterium comprises a mutation in cya and crp and further comprises a mutation in sopF. In some embodiments, a live intracellular delivery platform is provided wherein the attenuated Salmonella bacterium comprises a mutation in cya and crp and further comprises a mutation in feoABC. [00027] In some embodiments, a mutation is a deletion or an inactivation mutation. In embodiments, a mutation is a deletion mutation.
  • a vaccine comprising the attenuated Salmonella bacterium, or one or more attenuated Salmonella bacterium described herein.
  • an attenuated Salmonella bacterium is provided as a vaccine, vaccine component, immunogenic composition, or immunogen.
  • an immunogenic composition is provided comprising the attenuated Salmonella bacterium, or one or more attenuated Salmonella bacterium described herein.
  • the vaccine or immunogenic composition further comprises an adjuvant.
  • the attenuated Salmonella bacterium provides a mutant Salmonella bacterium capable of generating or stimulating an immune response in a host or animal, wherein the bacterium is avirulent in the host or animal.
  • the attenuated Salmonella bacterium fails to cause disease or significant pathology or illness in the host.
  • the attenuated Salmonella bacterium comprises a mutation in at least one of cya gene, crp gene, phoPQ gene, ompR-envZ gene, ssrAB gene, SPI2 gene, sifA gene, sseJ gene, sopF gene, SPI 13 gene, sitABCD gene, feoABC gene, mgtRBC gene, sspH2 gene.
  • the attenuated Salmonella bacterium comprises a mutation in at least one gene selected from cya, crp, phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2.
  • the attenuated Salmonella bacterium comprises a mutation in at least one of a cya and a crp gene.
  • the attenuated Salmonella bacterium comprises at least one first mutation and at least one second mutation; wherein the at least one first mutation is a mutation in a gene selected from cya and crp; and at least one second mutation is a mutation in a gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, asd, and aroA.
  • the attenuated Salmonella bacterium comprises at least one first mutation and at least one second mutation; wherein the at least one first mutation is selected from a mutation in cya and crp; and at least one second mutation is a mutation in a gene selected from phoPQ, ompR-envZ, sifA, sopF, feoABC.
  • the attenuated Salmonella bacterium comprises at least one first mutation and second mutation and at least one third mutation; wherein the at least one first mutation and second mutation is a mutation in a gene selected from cya and crp; and at least one third mutation is a mutation in a gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, asd, and aroA.
  • the attenuated Salmonella bacterium comprises at least one first mutation and at least one second mutation; wherein the at least one first mutation is selected from cya and crp; and at least one second mutation is selected from phoPQ, ompR-envZ, sifA, sopF, feoABC.
  • the attenuated Salmonella bacterium comprises at least one first mutation and second mutation and at least one third mutation; wherein the at least one first mutation and second mutation is selected from cya and crp; and at least one third mutation is selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, asd, and aroA.
  • the attenuated Salmonella bacterium comprises at least one first mutation and second mutation and at least one third mutation; wherein the at least one first mutation and second mutation is selected from cya and crp; and at least one third mutation is selected from phoPQ, ompR-envZ, sifA, sopF and feoABC.
  • the attenuated Salmonella bacterium comprises at least one first mutation and second mutation and at least one third mutation; wherein the at least one first mutation and second mutation is a mutation in a gene selected from cya and crp; and at least one third mutation is a mutation in a gene selected from phoPQ, ompR-envZ, sifA, sopF and feoABC.
  • the attenuated Salmonella bacterium comprises a first, second and third mutation; wherein the first mutation is a mutation in a gene for cya, the second mutation is a mutation in a gene for crp; and the third mutation is in a gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, asd, and aroA.
  • the attenuated Salmonella bacterium comprises a first, second and third mutation; wherein the first mutation is a mutation in a gene for cya, the second mutation is a mutation in a gene for crp; and the third mutation is a mutation in a gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, asd, and aroA.
  • the attenuated Salmonella bacterium comprises at least one first mutation and at least one second mutation; wherein the at least one first mutation is selected from cya and crp; and at least one second mutation is selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, asd, and aroA.
  • the attenuated Salmonella bacterium comprises at least one first mutation and at least one second mutation; wherein the at least one first mutation is a mutation in a gene selected from cya and crp; and at least one second mutation is a mutation in a gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, asd, and aroA.
  • the attenuated Salmonella bacterium comprises a first, second and third mutation; wherein the first mutation is a mutation in agene for cya, the second mutation is a mutation in a gene for crp; and the third mutation is in a gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sopF, feoABC, SPI 13 and sspH2.
  • the attenuated Salmonella bacterium comprises a first, second and third mutation; wherein the first mutation is a mutation in a gene for cya, the second mutation is a mutation in a gene for crp; and the third mutation is a mutation in a gene selected from phoPQ, ompR-envZ, sifA, and feoABC.
  • the attenuated Salmonella bacterium comprises a first, second and third mutation; wherein the first mutation is a mutation in a gene for cya, the second mutation is a mutation in a gene for crp; and the third mutation is a mutation in a gene selected from phoPQ, ompR-envZ, sifA, sopF and feoABC.
  • the attenuated Salmonella bacterium comprises a first, second and third mutation; wherein the first mutation is a mutation in a gene for cya, the second mutation is a mutation in a gene for crp; and the third mutation is in a gene selected from phoPQ, sifA and sopF.
  • the attenuated Salmonella bacterium comprises a first, second and third mutation; wherein the first mutation is a mutation in a gene for cya, the second mutation is a mutation in a gene for crp; and the third mutation is in a gene selected from phoPQ, ompR-envZ, sifA, sopF and sspH2.
  • combinations of attenuated Salmonella bacteria for example having distinct or non-overlapping mutations for attenuation, may be utilized as a live delivery platform or in one or more methods provided herein.
  • the distinct one or more attenuated Salmonella bacteria may express or deliver alternative therapeutics, antigens, antibodies or biomolecules.
  • the distinct one or more attenuated Salmonella bacteria may have alternative or unique growth or infectiveness characteristics, such as for example to deliver therapeutics, biomolecules, antigens, antibodies, etc at distinct timing or under distinct growth conditions in the target host or animal.
  • the attenuated Salmonella bacteria encodes and/or expresses a therapeutic molecule or biotherapeutic.
  • the therapeutic molecule or biotherapeutic is a heterologous therapeutic molecule or biotherapeutic, such as wherein the therapeutic molecule or biotherapeutic is non-native or is not natively encoded or expressed by or via the bacteria.
  • the attenuated Salmonella bacteria comprises nucleic acid encoding and/or expressing a therapeutic molecule or biotherapeutic.
  • the therapeutic molecule is encoded and/or expressed by integration in the attenuated Salmonella bacteria genome. In some embodiments, the therapeutic molecule is encoded and/or expressed via a plasmid or vector in the attenuated Salmonella bacteria. In some embodiments, the therapeutic molecule is encoded and/or expressed via a self-amplifying nucleic acid in the attenuated Salmonella bacteria. In some embodiments, the self-amplifying nucleic acid encodes for at least one alphavirus genome replication protein selected from nsP1, nsP2, nsP3, and nsP4.
  • the self- amplifying nucleic acid encodes for the alphavirus genome replication proteins nsP1, nsP2, nsP3, and nsP4. In some embodiments the self-amplifying nucleic acid is self-amplifying RNA.
  • the attenuated Salmonella bacteria encodes and/or expresses one or more therapeutic biomolecule, particularly a heterologous or a non-native therapeutic biomolecule. In some embodiments, the attenuated Salmonella bacteria encodes and/or expresses a biomolecule having a therapeutic effect selected from an antibody, a ribonucleic acid (RNA), a therapeutic peptide, an anti- infective peptide, and an antigen.
  • the attenuated Salmonella bacteria encodes and/or expresses a biomolecule having a therapeutic effect selected from an antibody, a ribonucleic acid (RNA), an anti-infective peptide, and an antigen.
  • the self-amplifying nucleic acid encodes one or more therapeutic biomolecule.
  • the self-amplifying nucleic acid encodes a biomolecule having a therapeutic effect selected from an antibody, a ribonucleic acid (RNA), a therapeutic peptide, an anti- infective peptide, and an antigen.
  • the self-amplifying nucleic acid encodes a biomolecule having a therapeutic effect selected from an antibody, a ribonucleic acid (RNA), an anti- infective peptide, and an antigen.
  • the biomolecule having a therapeutic effect is an antibody.
  • the biomolecule having a therapeutic effect is an antibody fragment.
  • the biomolecule having a therapeutic effect is a single domain antibody.
  • the biomolecule having a therapeutic effect is an antibody fragment wherein the fragment consists of the variable heavy chain domain.
  • the biomolecule having a therapeutic effect is a camelid or VHH antibody.
  • the biomolecule is an antibody and is directed against a bacterial toxin.
  • the biomolecule is a camelid antibody and is directed against a bacterial toxin.
  • the biomolecule is a camelid antibody is directed against a toxin of Clostridium perfringens.
  • the camelid antibody comprises SEQ ID NO: 169.
  • the biomolecule having a therapeutic effect is a small hairpin RNA (shRNA) or a short interfering RNA (siRNA).
  • shRNA or siRNA comprises SEQ ID NO: 135-146.
  • the shRNA comprises or is selected from SEQ ID NO: 135-138.
  • the siRNA comprises or is selected from SEQ ID NO: 139-146.
  • the biomolecule having a therapeutic effect is an antigen.
  • the biomolecule having a therapeutic effect is an antigen derived from an infective agent or a disease causing agent.
  • the biomolecule having a therapeutic effect is a bacterial antigen.
  • the biomolecule having a therapeutic effect is a viral antigen.
  • the biomolecule having a therapeutic effect is a parasite antigen.
  • the biomolecule having a therapeutic effect is an antigen derived from an Eimeria parasite.
  • the Eimeria antigen is selected from Eimeria tenella elongation factor - 1 ⁇ ; EtAMA1; Eimeria tenella 5401; Eimeria acervulina lactate dehydrogenase antigen gene; Eimeria maxima surface antigen gene; Glyceraldehyde 3-phosphate Dehydrogenase (GAPDH); Eimeria common antigen 14-3-3.
  • the live delivery platform comprises an Eimeria antigen selected from Eimeria tenella elongation factor -1 ⁇ ; EtAMA1; Eimeria tenella 5401; Eimeria acervulina lactate dehydrogenase antigen gene; Eimeria maxima surface antigen gene; Glyceraldehyde 3-phosphate Dehydrogenase (GAPDH); Eimeria common antigen 14-3-3, or an antigenic fragment or portion thereof.
  • the live delivery platform comprises an Eimeria antigen protein selected from EtAMA1; Eimeria tenella 5401, Glyceraldehyde 3-phosphate Dehydrogenase (GAPDH) and Eimeria common antigen 14-3-3, or an antigenic fragment or portion thereof.
  • the Eimeria antigen is selected from the antigen encoding nucleic acid as set out in any of SEQ ID NO: 153-157 or 173-177. In some embodiments, the Eimeria antigen is selected from GAPDH, 14-3-3 and AMA1. [00044] In embodiments, the Eimeria antigen is a portion of the Eimeria protein which is sufficient to generate protective antibodies and/or to provide mucosal, systemic and cellular immunity against Eimeria. Thus, it is contemplated that the entire antigen protein may not need to be expressed. A sufficient antigenic portion or region of the protein sufficient to provide antibody response and immunity in an animal is required. In some embodiments, an antigenic fragment of the protein is expressed.
  • the antigenic portion or fragment is a non-naturally occurring fragment of the protein.
  • a live intracellular delivery platform comprising: an attenuated Salmonella bacterium having mutations in a cya gene and crp gene; and nucleic acid encoding and/or expressing a therapeutic biomolecule, particularly a heterologous therapeutic biomolecule.
  • a live intracellular delivery platform comprising: an attenuated Salmonella bacterium having mutations in cya gene and crp gene, wherein the bacterium additionally comprises one or mutation in a Salmonella gene selected from phoPQ, ompR- envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, aroA, and thyA; and nucleic acid encoding and/or expressing a therapeutic biomolecule, particularly a heterologous therapeutic biomolecule.
  • a live intracellular delivery platform comprising: an attenuated Salmonella bacterium having mutations in cya gene and crp gene, wherein the bacterium additionally comprises one or mutation in a Salmonella gene selected from phoPQ, ompR- envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, asd and thyA; and nucleic acid encoding and/or expressing a therapeutic biomolecule, particularly a heterologous therapeutic biomolecule.
  • a live intracellular delivery platform comprising: an attenuated Salmonella bacterium having mutations in cya gene and crp gene, wherein the bacterium further comprises one or mutation in a Salmonella gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, sspH2 and mgtRBC; and nucleic acid encoding and/or expressing a therapeutic biomolecule, particularly a heterologous therapeutic biomolecule.
  • a live intracellular delivery platform comprising: an attenuated Salmonella bacterium having mutations in cya gene and crp gene, wherein the bacterium additionally comprises one or mutation in a Salmonella gene selected from phoPQ, ompR- envZ, sifA, sopF, feoABC, and ssPH2; and nucleic acid encoding and/or expressing a therapeutic biomolecule, particularly a heterologous therapeutic biomolecule.
  • a live intracellular delivery platform comprising: an attenuated Salmonella bacterium having mutations in cya gene and crp gene, wherein the bacterium additionally comprises one or mutation in a Salmonella gene selected from phoPQ, ompR- envZ, sifA, sopF and ssPH2; and nucleic acid encoding and/or expressing a therapeutic biomolecule, particularly a heterologous therapeutic biomolecule.
  • a live intracellular delivery platform comprising: an attenuated Salmonella bacterium having mutations in cya gene and crp gene, wherein the bacterium additionally comprises one or mutation in a Salmonella gene selected from phoPQ, ompR- envZ, sifA, sopF and feoABC; and nucleic acid encoding and/or expressing a therapeutic biomolecule, particularly a heterologous therapeutic biomolecule.
  • a live intracellular delivery platform comprising: an attenuated Salmonella bacterium having mutations in cya gene and crp gene, wherein the bacterium additionally comprises one or mutation in a Salmonella gene selected from phoPQ, sifA and sopF; and nucleic acid encoding and/or expressing a therapeutic biomolecule, particularly a heterologous therapeutic biomolecule.
  • a live intracellular delivery platform comprising: an attenuated Salmonella bacterium having mutations in cya gene and crp gene; and self amplifying RNA that encodes for proteins nsP1, nsP2, nsP3, and nsP4, and a therapeutic biomolecule.
  • a live intracellular delivery platform comprising: an attenuated Salmonella bacterium having mutations in cya gene and crp gene, wherein the bacterium additionally comprises one or mutation in a Salmonella gene selected from phoPQ, ompR- envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, aroA, and thyA; and self amplifying RNA that encodes for proteins nsP1, nsP2, nsP3, and nsP4, and a therapeutic biomolecule.
  • a Salmonella gene selected from phoPQ, ompR- envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba
  • a live intracellular delivery platform comprising: an attenuated Salmonella bacterium having mutations in cya gene and crp gene, wherein the bacteria additionally comprises one or mutation in a Salmonella gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, asd and thyA; and self amplifying RNA that encodes for proteins nsP1, nsP2, nsP3, and nsP4, and a therapeutic biomolecule.
  • a live intracellular delivery platform comprising: attenuated Salmonella bacterium having mutations in cya gene and crp gene, wherein the bacteria additionally comprises one or mutation in a Salmonella gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, sspH2 and mgtRBC; and self amplifying RNA that encodes for proteins nsP1, nsP2, nsP3, and nsP4, and a therapeutic biomolecule.
  • a live intracellular delivery platform comprising: attenuated Salmonella bacterium having mutations in cya gene and crp gene, wherein the bacteria additionally comprises one or mutation in a Salmonella gene selected from phoPQ, ompR-envZ, sifA, sopF, feoABC, and ssPH2; and self amplifying RNA that encodes for proteins nsP1, nsP2, nsP3, and nsP4, and a therapeutic biomolecule.
  • a live intracellular delivery platform comprising: attenuated Salmonella bacterium having mutations in cya gene and crp gene, wherein the bacteria additionally comprises one or mutation in a Salmonella gene selected from phoPQ, ompR-envZ, sifA, sopF and ssPH2; and self amplifying RNA that encodes for proteins nsP1, nsP2, nsP3, and nsP4, and a therapeutic biomolecule.
  • a live intracellular delivery platform comprising: attenuated Salmonella bacterium having mutations in cya gene and crp gene, wherein the bacteria additionally comprises one or mutation in a Salmonella gene selected from phoPQ, ompR-envZ, sifA, sopF and feoABC; and self amplifying RNA that encodes for proteins nsP1, nsP2, nsP3, and nsP4, and a therapeutic biomolecule.
  • a live intracellular delivery platform comprising: attenuated Salmonella bacterium having mutations in cya gene and crp gene, wherein the bacteria additionally comprises one or mutation in a Salmonella gene selected from phoPQ, sifA and sopF; and self amplifying RNA that encodes for proteins nsP1, nsP2, nsP3, and nsP4, and a therapeutic biomolecule.
  • an attenuated Salmonella bacterium having mutations in the cya gene and crp gene, wherein the bacterium additionally comprises one or mutation in a Salmonella gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, aroA, and thyA.
  • a Salmonella gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, aroA, and thyA.
  • an attenuated Salmonella bacterium having mutations in the cya gene or crp gene, wherein the bacterium additionally comprises one or mutation in a Salmonella gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, aroA, and thyA.
  • a Salmonella gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, aroA, and thyA.
  • an attenuated Salmonella bacterium comprising a mutation in a cya gene and/or a crp gene, further comprising one or mutation in a Salmonella gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, aroA, and thyA.
  • an attenuated Salmonella bacterium comprising a mutations in a cya gene and a crp gene, wherein the bacterium additionally comprises one or mutation in a Salmonella gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, aroA, and thyA.
  • an attenuated Salmonella bacterium comprising a mutation in a cya gene and/or a crp gene, further comprising one or mutation in a Salmonella gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, and aroA.
  • an attenuated Salmonella bacterium comprising mutations in a cya gene and a crp gene, wherein the bacterium additionally comprises one or mutation in a Salmonella gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, and aroA.
  • an attenuated Salmonella bacterium comprising a mutation in a cya gene and/or a crp gene, and further comprising a mutation in at least one gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, and aroA.
  • an attenuated Salmonella bacterium comprising a mutation in a cya and a crp gene, and further comprising a mutation in at least one gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, and aroA.
  • an attenuated Salmonella bacterium having mutations in a Salmonella gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, aroA, and thyA.
  • an attenuated Salmonella bacterium is provided having mutations in a Salmonella gene selected from phoPQ, ompR-envZ, sifA, sopF and feoABC.
  • an attenuated Salmonella bacterium comprising a mutation in at least one gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, aroA, and thyA.
  • an attenuated Salmonella bacterium comprising a mutation in at least one gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, and aroA.
  • an attenuated Salmonella bacterium is provided comprising a mutation in at least one gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sopF, SPI 13, feoABC, and sspH2.
  • an attenuated Salmonella bacterium having mutations in a Salmonella gene selected from phoPQ, ompR-envZ, sifA, sopF and feoABC.
  • an attenuated Salmonella bacterium comprising a mutation in a crp and cya gene and further comprising a mutation at least one gene selected from phoPQ, ompR- envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, aroA, and thyA.
  • an attenuated Salmonella bacterium comprising a mutation in a crp and cya gene and further comprising a mutation in at least one gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, and aroA.
  • an attenuated Salmonella bacterium comprising a mutation in a crp and cya gene and further comprising a mutation in at least one gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sopF, SPI 13, feoABC, and sspH2.
  • an attenuated Salmonella bacterium is provided having mutations in a Salmonella gene selected from phoPQ, ompR-envZ, sifA, sopF and feoABC.
  • the attenuated Salmonella bacterium further comprises a mutation in a crp and cya gene.
  • an attenuated Salmonella bacterium having mutations in the cya gene and crp gene, wherein the bacterium additionally comprises one or mutation in a Salmonella gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sopF, feoABC, SPI 13 and ssPH2.
  • an attenuated Salmonella bacterium is provided having mutations in the cya gene and crp gene, wherein the bacterium additionally comprises one or mutation in a Salmonella gene selected from phoPQ, ompR-envZ, sifA, and feoABC.
  • an attenuated Salmonella bacterium having mutations in the cya gene and crp gene, wherein the bacterium additionally comprises a mutation in a Salmonella gene phoPQ. In embodiments, an attenuated Salmonella bacterium having mutations in the cya gene and crp gene, wherein the bacterium additionally comprises a mutation in a Salmonella gene ompR-envZ. In embodiments, an attenuated Salmonella bacterium is provided having mutations in the cya gene and crp gene, wherein the bacterium additionally comprises a mutation in a Salmonella gene sifA.
  • an attenuated Salmonella bacterium having mutations in the cya gene and crp gene, wherein the bacterium additionally comprises a mutation in a Salmonella gene feoABC. In embodiments, an attenuated Salmonella bacterium is provided having mutations in the cya gene and crp gene, wherein the bacterium additionally comprises a mutation in a Salmonella gene sopF. In some embodiments, attenuated Salmonella bacterium having mutations in the cya gene and crp gene is provided, wherein the bacterium additionally comprises one or mutation in a Salmonella gene selected from phoPQ, ompR-envZ, sifA, sopF and ssPH2.
  • an attenuated Salmonella bacterium having mutations in the cya gene and crp gene, wherein the bacterium additionally comprises one or mutation in a Salmonella gene selected from phoPQ, ompR-envZ, sifA, sopF and feoABC.
  • an attenuated Salmonella bacterium is provided having mutations in the cya gene and crp gene, wherein the bacterium additionally comprises one or mutation in a Salmonella gene selected from phoPQ, sifA and sopF.
  • the mutation in one or more gene is a deletion of at least a portion of the gene or genes resulting in inactivation of the gene or genes.
  • Attenuated Salmonella bacterium comprising a self-amplifying nucleic acid for use in therapy.
  • an attenuated Salmonella bacterium comprising a self-amplifying nucleic acid for use in therapy, wherein the attenuated Salmonella bacterium comprises a mutation at least one gene selected from cya, crp, phoPQ, ompR- envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd and aroA.
  • an attenuated Salmonella bacterium having mutations in the cya gene and crp gene, wherein the bacterium additionally comprises one or mutation in a Salmonella gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, aroA, and thyA comprising a self-amplifying nucleic acid for use in therapy.
  • a Salmonella gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, aroA, and thyA comprising a self-amplifying nucleic acid for use in therapy
  • Attenuated Salmonella bacterium having mutations in the cya gene or crp gene, wherein the bacterium additionally comprises one or mutation in a Salmonella gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, aroA, and thyA comprising a self-amplifying nucleic acid for use in therapy.
  • a Salmonella gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, aroA, and thyA comprising a self-amplifying nucleic acid for use in therapy.
  • an attenuated Salmonella bacterium having mutations in a Salmonella gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, aroA, and thyA comprising a self-amplifying nucleic acid for use in therapy.
  • an attenuated Salmonella bacterium comprising a self- amplifying nucleic acid for use in manufacture of a medicament to treat a disease or disorder modulated by an antibody or a ribonucleic acid (RNA), wherein the attenuated Salmonella bacterium comprises a mutation in at least one gene selected from cya, crp, phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, and aroA.
  • RNA ribonucleic acid
  • an attenuated Salmonella bacterium comprising a self- amplifying nucleic acid for use in manufacture of a medicament to elicit immunity in a non-human animal, wherein the self-replicating nucleic acid encodes an antigen, wherein the attenuated Salmonella bacterium comprises a mutation in at least one gene selected from cya, crp, phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, and aroA.
  • an attenuated Salmonella bacterium comprising a self- amplifying nucleic acid for use in manufacture of a medicament to treat or prevent infection of a non- human animal by a pathogen, wherein the attenuated Salmonella bacterium comprises a mutation in at least one gene selected from cya, crp, phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, and aroA and wherein the self-amplifying nucleic acid encodes an anti-infective peptide.
  • a live intracellular delivery platform comprising: attenuated Salmonella bacterium having mutations in a cya gene and a crp gene and a mutation in at least one gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, and sspH2; and a nucleic acid that encodes and expresses a heterologous therapeutic biomolecule.
  • the nucleic acid is self-amplifying nucleic acid.
  • Attenuated Salmonella bacterium having mutations in a Salmonella gene selected from phoPQ, ompR-envZ, sifA, sopF and feoABC comprising a self- amplifying nucleic acid for use in therapy.
  • attenuated Salmonella bacterium is provided having mutations in a Salmonella gene selected from phoPQ, sifA and sopF comprising a self-amplifying nucleic acid for use in therapy.
  • an attenuated Salmonella bacterium is provided comprising a self- amplifying nucleic acid for use in manufacture of a medicament to treat a disease or disorder.
  • an attenuated Salmonella bacterium comprising a self-amplifying nucleic acid for use in manufacture of a medicament to treat a disease or disorder modulated by an antibody or a ribonucleic acid (RNA).
  • an attenuated Salmonella bacterium is provided comprising a self-amplifying nucleic acid for use in manufacture of a medicament to treat a disease or disorder modulated by an antibody.
  • an attenuated Salmonella bacterium is provided comprising a self-amplifying nucleic acid for use in manufacture of a medicament to treat a disease or disorder caused by an infectious agent or pathogen.
  • Attenuated Salmonella bacterium comprising a self- amplifying nucleic acid for use in manufacture of a medicament to elicit immunity in a non-human animal, wherein the self-replicating nucleic acid encodes an antigen.
  • attenuated Salmonella bacterium is provided comprising a self- amplifying nucleic acid for use in manufacture of a medicament to treat or prevent infection of a non- human animal by a pathogen, wherein the self-amplifying nucleic acid encodes an anti-infective peptide.
  • Attenuated Salmonella bacterium comprising a self-amplifying nucleic acid for use in manufacture of a medicament to treat or prevent infection of a non-human animal by an infectious agent, wherein the self-amplifying nucleic acid encodes an anti-infective peptide.
  • attenuated Salmonella bacterium is provided comprising a self-amplifying nucleic acid for use in manufacture of a medicament to treat or prevent infection of a non-human animal by a bacteria, wherein the self-amplifying nucleic acid encodes an anti-bacterial peptide or a bacterial antigen.
  • Attenuated Salmonella bacterium comprising a self- amplifying nucleic acid for use in manufacture of a medicament to treat or prevent infection of a non- human animal by a bacteria, wherein the self-amplifying nucleic acid encodes an bacterial lytic polypeptide or lysin.
  • a self-amplifying nucleic acid comprising sequences encoding alphavirus genome replication proteins nsP1, nsP2, nsP3, and nsP4; and sequences encoding a biomolecule having a therapeutic effect.
  • a self-amplifying nucleic acid comprising sequences encoding alphavirus genome replication proteins nsP1, nsP2, nsP3, and nsP4.
  • sequences encoding a biomolecule having a therapeutic effect are selected from the group consisting of an antibody, a ribonucleic acid (RNA), an anti-infective peptide and an antigen.
  • the invention includes a method for intracellular delivery of a therapeutic biomolecule comprising: (a) contacting an attenuated Salmonella bacterium with a cell, wherein the attenuated Salmonella bacterium comprises a self-amplifying nucleic acid that encodes for a therapeutic biomolecule; (b) lysing the attenuated Salmonella bacterium in a cell; and (c) synthesizing the therapeutic biomolecule by a cell.
  • a method for intracellular delivery of a therapeutic biomolecule comprising: (a) contacting an attenuated Salmonella bacterium with a cell, wherein the attenuated Salmonella bacterium comprises a nucleic acid that encodes and/or expresses a therapeutic biomolecule, particularly a heterologous therapeutic biomolecule; (b) lysing the attenuated Salmonella bacterium in the cell; and (c) synthesizing the therapeutic biomolecule by the cell.
  • nucleic acid encoding the therapeutic molecule is retained in the cell and the therapeutic biomolecule is synthesized thereby in the cell or by the cell.
  • a method for intracellular delivery of a therapeutic biomolecule comprising: (a) contacting an attenuated Salmonella bacterium with a cell, wherein the attenuated Salmonella bacterium comprises a nucleic acid that encodes and/or expresses a therapeutic biomolecule, particularly a heterologous therapeutic biomolecule; (b) wherein the attenuated Salmonella bacterium is then lysed in the cell; and (c) wherein the therapeutic biomolecule is synthesized by or in the cell.
  • the attenuated Salmonella bacterium comprises a mutation in cya gene and crp gene.
  • the attenuated Salmonella bacterium comprises a mutation in a Salmonella gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, aroA, and thyA.
  • the attenuated Salmonella bacterium comprises a mutation in a Salmonella gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, and aroA.
  • the attenuated Salmonella bacterium comprises a mutation in a Salmonella gene selected from phoPQ, ompR-envZ, sifA, sopF, feoABC, and ssPH2.
  • the attenuated Salmonella bacterium comprises a mutation in a Salmonella gene selected from phoPQ, ompR-envZ, sifA, sopF and feoABC. In some embodiments of the method, the attenuated Salmonella bacterium comprises a mutation in a Salmonella gene selected from phoPQ, sifA and sopF. [00086] In some embodiments of the method, the self-amplifying nucleic acid encodes for at least one alphavirus genome replication protein selected from nsP1, nsP2, nsP3, and nsP4.
  • the self-amplifying nucleic acid encodes for the alphavirus genome replication proteins nsP1, nsP2, nsP3, and nsP4. In some embodiments, the self-amplifying nucleic acid encodes for the alphavirus genome replication proteins nsP1, nsP2, nsP3, and nsP4 and the structural proteins of the alphavirus. [00087] In some embodiments of the method, the cell of (a) is an M cell. In some embodiments of the method, the cell of (c) is a macrophage or enterocyte or epithelial cell.
  • the therapeutic biomolecule is selected from: antibody, a ribonucleic acid (RNA), an anti-infective peptide, and an antigen.
  • the therapeutic biomolecule comprises an Eimeria parasite antigen.
  • the therapeutic biomolecule is selected from Glyceraldehyde 3-phosphate Dehydrogenase (GAPDH) and Eimeria common antigen 14-3-3.
  • a method for intracellular delivery of a therapeutic biomolecule comprising contacting an attenuated Salmonella bacterium with a cell, wherein the attenuated Salmonella bacterium comprises a self-amplifying nucleic acid that encodes for a therapeutic biomolecule, wherein the bacteria is killed in the cell and the self-amplifying nucleic acid is retained in the cell and synthesizes the therapeutic biomolecule in the cell.
  • a method for systemic delivery of a therapeutic biomolecule comprising: (a) contacting attenuated Salmonella bacterium with a cell, wherein the attenuated Salmonella bacterium comprises a nucleic acid that encodes for and is capable of expressing a therapeutic biomolecule, particularly a heterologous therapeutic biomolecule; (b) lysing the attenuated Salmonella bacterium in the cell; (c) synthesizing the therapeutic biomolecule in the cell; and (d) circulating the therapeutic biomolecule synthesized by the cell of step (c).
  • a method for systemic delivery of a therapeutic biomolecule in an animal comprising: (a) contacting attenuated Salmonella bacterium with a cell, wherein the attenuated Salmonella bacterium comprises a nucleic acid that encodes for and is capable of expressing a therapeutic biomolecule, particularly a heterologous therapeutic biomolecule; (b) lysing the attenuated Salmonella bacterium in the cell; (c) synthesizing the therapeutic biomolecule in the cell; and (d) circulating the therapeutic biomolecule synthesized by the cell of step (c) in the animal.
  • the synthesized therapeutic molecule is circulated in the animal via the intestinal system.
  • the synthesized therapeutic molecule is circulated in the animal via the blood stream.
  • a method for systemic delivery of a therapeutic biomolecule comprising: (a) contacting attenuated Salmonella bacterium with a cell, wherein the attenuated Salmonella bacterium comprises a self-amplifying nucleic acid that encodes for a therapeutic biomolecule; (b) lysing the attenuated Salmonella bacterium in the cell; (c) synthesizing the therapeutic biomolecule in the cell; and (d) circulating the therapeutic biomolecule synthesized by the cell of step (c).
  • the attenuated Salmonella bacterium comprises a mutation in cya gene and crp gene.
  • the attenuated Salmonella bacterium comprises a mutation in a Salmonella gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, aroA, and thyA.
  • the attenuated Salmonella bacterium comprises a mutation in a Salmonella gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd and aroA.
  • the attenuated Salmonella bacterium comprises a mutation in a Salmonella gene selected from phoPQ, ompR-envZ, sifA, sopF, feoABC, and ssPH2.
  • the nucleic acid is self-amplifying nucleic acid.
  • the self-amplifying nucleic acid encodes for at least one alphavirus genome replication protein selected from nsP1, nsP2, nsP3, and nsP4.
  • the self- amplifying nucleic acid encodes for the alphavirus genome replication proteins nsP1, nsP2, nsP3, and nsP4.
  • the self-amplifying nucleic acid encodes for the alphavirus genome replication proteins nsP1, nsP2, nsP3, and nsP4 and the structural proteins of the alphavirus.
  • the cell of (a) is an M cell. In some embodiments of the method, the cell of (c) is a macrophage or enterocyte or epithelial cell.
  • the therapeutic biomolecule is selected from antibody, a ribonucleic acid (RNA), an anti-infective peptide, and an antigen.
  • the therapeutic biomolecule comprises an Eimeria parasite antigen. In some embodiments of the method, the therapeutic biomolecule comprises an antigen selected from Glyceraldehyde 3-phosphate Dehydrogenase (GAPDH) and Eimeria common antigen 14-3-3.
  • GPDH Glyceraldehyde 3-phosphate Dehydrogenase
  • a method for systemic delivery of a therapeutic biomolecule comprising contacting an attenuated Salmonella bacterium with a cell, wherein the attenuated Salmonella bacterium comprises a nucleic acid that encodes for a therapeutic biomolecule, particularly a heterologous therapeutic biomolecule, wherein the bacteria is killed in the cell and the nucleic acid is retained in the cell and the therapeutic biomolecule is synthesized or retained in the cell and the therapeutic biomolecule is released outside of the cell in an animal.
  • a method for systemic delivery of a therapeutic biomolecule comprising contacting an attenuated Salmonella bacterium with a cell, wherein the attenuated Salmonella bacterium comprises a self-amplifying nucleic acid that encodes for a therapeutic biomolecule, wherein the bacteria is killed in the cell and the self-amplifying nucleic acid is retained in the cell and synthesizes the therapeutic biomolecule in the cell and the therapeutic biomolecule is released outside of the cell in an animal.
  • FIGURE 1A provides schematic representation of a self-amplifying nucleic acid.
  • the genomic RNA from an alphavirus is modified to express a gene of interest from a subgenomic promoter.
  • FIGURE 1B depicts the A69 plasmid (13,291 bp).
  • the GFP cassette is introduced as a gene of interest to generate the A69 plasmid (13,291 bp).
  • Expression of the nonstructural proteins (nsPs) are directed by a CMV promoter, whereas GFP is expressed by a subgenomic alphavirus promoter.
  • FIGURE 2 depicts expression of GFP using the SAM-RNA platform.
  • FIGURE 3A provides a schematic representation of chromosomal deletion of Salmonella phagocyte survival factors.
  • FIGURE 3B provides agarose gel electrophoresis images of confirmation of gene deletions in S. Typhimurium UK1 ⁇ crp-cya derivatives. PCR amplification was performed to confirm the deletions.
  • the primers were designed flanking the genes of interest.
  • FIGURE 4A and 4B provides a comparison of growth kinetics of various survival factor Salmonella bacteria mutants.
  • FIGURE 4A provides growth curves and depicts time (hours) and OD600.
  • FIGURE 4B provides growth rate in doubling time, calculated from the OD values.
  • FIGURE 5 Survival of mutant bacteria inside macrophage host cells. The fold difference versus MeganVac is presented.
  • FIGURE 6A provides fluorescent images for each deletion mutant phoPQ, ompR, ssrAB, SPI2, sifA, sseJ, sopF, SP113, sitABCD, feoABC, and mgtRBC strain and MeganVac transformed with GFP encoding plasmid A69, as well as the untransformed MeganVac strain.
  • FIGURE 6B provides brightfield images for each deletion mutant phoPQ, ompR, ssrAB, SPI2, sifA, sseJ, sopF, SP113, sitABCD, feoABC, and mgtRBC strain and MeganVac transformed with GFP encoding plasmid A69, as well as the untransformed MeganVac strain.
  • FIGURE 7. GFP tagged VHH expression from SAM vector versus a DNA vector plasmid CI. Control is the pCI plasmid vector expressing GFP alone (maxGFP).
  • FIGURE 8 siRNA and shRNA mediated knockdown of CD163/SRCR5 in a swine macrophage cell line.
  • FIGURE 9 shows SAM-mediated expression of GAPDH, AMA1 and 14-3-3 proteins.
  • FIGURE 10 provides a graphical representation of the major Salmonella genes upregulated inside macrophages and their environmental cues.
  • FIGURE 11 provides a schematic diagram showing the construction of Salmonella typhimurium mutants.
  • FIGURE 12 depicts in vitro growth characteristic of Salmonella mutants in defined media.
  • A growth curve and
  • B doubling time of mutants in M9 medium supplemented with 0.1% glucose and 0.12% casamino acid. Designated mutants are indicated.
  • FIGURE 13 depicts in vitro growth characteristic of Salmonella mutants in macrophage- simulated media. (A) growth curve and (B) doubling time of mutants in PCN media. Designated mutants are indicated.
  • FIGURE 14 provides macrophage survival assay of Salmonella mutants inside HD11 macrophage cell line and particularly invasion (A) and intra-macrophage survival (B) of S.
  • FIGURE 15 depicts confirmation of differentiation of chicken PBMCs into primary macrophages.
  • PBMCs were isolated from the heparinized blood collected from healthy birds and separated using Histopaque 1077.
  • A Phase-contrast images of cultured chicken PBMCs at different time points.
  • B Flow cytometric characterization of chicken macrophages. Cells were stained with chicken macrophage-specific antibody KUL01.
  • C Infection of chicken primary macrophages with MeganVac1 ( ⁇ crp ⁇ cya) cells carrying a mCherry expression plasmid. Note the formation of SCVs and the presence of Salmonella inside SCVs.
  • FIGURE 16 provides Venn diagrams in each of A, B, C and D comparing the DEGs between UK-1, MeganVac1, phoPQ, sifA and sopF mutants.
  • A compares UK1 and MeganVac1
  • B compares UK-1, MeganVac1 and phoPQ
  • C compares UK-1, MeganVac1 and sifa
  • D compares UK-1, MeganVac1 and sopF.
  • FIGURE 17 provides a comparison of the gene expression in S. Typhimurium UKl, ⁇ crp- ⁇ cya (MeganVac1) and its mutant derivatives.
  • FIGURE 18 provides a schematic diagram of the response of macrophages to Salmonella invasion.
  • DETAILED DESCRIPTION [000125]
  • subject includes bird, poultry, a human, or a non-human animal. Specific examples include chickens, turkey, dogs, cats, cattle, and swine. The chicken may be a broiler chicken, egg-laying or egg-producing chicken.
  • RNA or DNA RNA or DNA
  • a "heterologous" region of a nucleic acid, RNA or DNA, construct is an identifiable segment of RNA or DNA within a larger RNA or DNA molecule that is not found in association with the larger molecule in nature.
  • the heterologous region encodes a gene
  • the gene will usually be flanked by RNA or DNA that does not flank the genomic RNA or DNA in the genome of the source organism.
  • primer refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH.
  • the primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent.
  • the exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
  • the primers herein are selected to be "substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand.
  • a "chimeric protein” or “fusion protein” comprises all or (preferably a biologically active) part of a first polypeptide operably linked to a heterologous polypeptide. Chimeric proteins or peptides are produced, for example, by combining two or more proteins having two or more active sites.
  • a first polypeptide may be covalently attached to an entity which may provide additional function or enhance the use or application of the first polypeptide(s), including for instance a tag, label, targeting moiety or ligand, a cell binding or cell recognizing motif or agent, an antibacterial agent, an antibody, an antibiotic.
  • exemplary labels include a radioactive label, such as the isotopes 3 H, 14 C, 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, and 186 Re.
  • the label may be an enzyme, and detection of the labeled lysin polypeptide may be accomplished by any of the presently utilized or accepted colorimetric, spectrophotometric, fluorospectrophotometric, amperometric, or gasometric techniques known in the art. Chimeric protein and peptides can act independently on the same or different molecules or targets, and hence have a potential to provide multiple activities, such as to treat or stimulate immune response against two or more different bacterial infections or infective agents at the same time.
  • expression inhibitory agent or ‘expression inhibiting agent’ means an agent, e.g.
  • ‘expression inhibitory agent’ comprises a DNA or RNA molecule that contains a nucleotide sequence identical to or complementary to at least about 15-30, particularly at least 17, sequential nucleotides within the polyribonucleotide sequence coding for a specific polypeptide or protein.
  • Exemplary such expression inhibitory molecules include ribozymes, microRNAs, double stranded siRNA molecules, self-complementary single-stranded siRNA molecules, shRNA, genetic antisense constructs, and synthetic RNA antisense molecules with modified stabilized backbones.
  • microRNA or "miRNA” or “miR” as used herein refers to its meaning as is generally accepted in the art. More specifically, the term refers a small double-stranded RNA molecules that regulate the expression of target messenger RNAs either by mRNA cleavage, translational repression/inhibition or heterochromatic silencing (see for example Ambros, 2004, Nature, 431 , 350-355; Barrel, 2004, Cell, 116, 281-297; Cullen, 2004, Virus Research., 102, 3-9; He et al, 2004, Nat. Rev.
  • RNA includes mature single stranded miRNAs, precursor miRNAs (pre-miR), and variants thereof, which may be naturally occurring.
  • miRNA also includes primary miRNA transcripts and duplex miRNAs.
  • a particular inhibitory agent is a small interfering RNA (siRNA, particularly small hairpin RNA, “shRNA”).
  • siRNA mediate the post-transcriptional process of gene silencing by double stranded RNA (dsRNA) that is homologous in sequence to the silenced RNA.
  • siRNA according to aspects provided herein comprises a sense strand of 15-30, particularly 17-30, most particularly 17-25 nucleotides complementary or homologous to a contiguous 17-25 target nucleotide sequence, and an antisense strand of 15-30, particularly 17-30, most particularly 17-25, more specifically 19-21 nucleotides complementary to the sense strand. More particular siRNA according to aspects provided herein comprises a sense strand selected from the group of sequences comprising SEQ ID NOs: 135-138.
  • the most particular siRNA comprises sense and anti-sense strands that are 100 per cent complementary to each other and the target polynucleotide sequence.
  • the siRNA further comprises a loop region linking the sense and the antisense strand.
  • a self-complementing single stranded shRNA molecule polynucleotide comprises a sense portion and an antisense portion connected by a loop region linker.
  • the loop region sequence is 4-30 nucleotides long, more particularly 5-15 nucleotides long and most particularly 8 or 12 nucleotides long.
  • Self-complementary single stranded siRNAs form hairpin loops and are more stable than ordinary dsRNA. In addition, they are more easily produced from vectors.
  • miRNAs are small non-coding RNAs, belonging to a class of regulatory molecules found in many eukaryotic species that control gene expression by binding to complementary sites on target messenger RNA (mRNA) transcripts.
  • miRNAs have been shown to regulate gene expression in two ways. First, miRNAs binding to protein-coding mRNA sequences that are exactly complementary to the miRNA induce the RNA-mediated interference (RNAi) pathway. Messenger RNA targets are cleaved by ribonucleases in the RISC complex.
  • RNAi RNA-mediated interference
  • miRNAs that bind to imperfect complementary sites on messenger RNA transcripts direct gene regulation at the posttranscriptional level but do not cleave their mRNA targets. miRNAs identified in both plants and animals use this mechanism to exert translational control over their gene targets.
  • mutations include point mutations, insertions, and deletions.
  • a deletion includes deletion of a part or entire gene.
  • Such mutations may have functional effects such as, for example, a decrease in function of a gene product, ablation of function in a gene product, and/or a new or altered function in a gene product.
  • the terms “treating”, “to treat”, or “treatment”, include restraining, slowing, stopping, reducing, ameliorating, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.
  • a treatment may be applied prophylactically or therapeutically.
  • “vaccine” refers to a composition that improves immunity to a particular disease.
  • a vaccine typically contains an agent that resembles a disease-causing pathogen, and is often made from weakened or killed forms of the pathogen, its toxins or one of its surface proteins.
  • immunogenic means than an agent is capable of eliciting an immune response, including an innate, humoral, or cellular immune response, and both. “Immunogenic” includes “immunomodulatory”.
  • An immunogenic composition is a composition that elicits an innate, humoral, or cellular immune response, or both.
  • the term “immune response” includes a response by a subject that involves generation of antibodies that bind to an antigen (i.e., an antibody response).
  • the phrase “stimulating an immune response” includes a) generating an immune response against an antigen (e.g., a viral antigen) in a na ⁇ ve individual; or b) increasing, reconstituting, boosting, or maintaining an immune response in an individual beyond what would occur if the composition was not administered.
  • a composition is immunogenic if it is capable of attaining either of these criteria when administered in single or multiple doses.
  • an immune or immunological response refers to administration of a composition that initiates, boosts, modulates, or maintains the capacity for the host's immune system to react to a virus or antigen, at a level higher than would otherwise occur.
  • adjuvant(s) describes a substance, compound, agent or material useful for improving an immune response or immune cell or component stimulation, and may in some instances be combined with any particular antigen in an immunological, pharmaceutical or vaccine composition. Adjuvants can be used to increase the amount of antibody and effector T cells produced and to reduce the quantity of antigen or immune stimulant or modulator and the frequency of injection.
  • an adjuvant is physiologically and/or pharmaceutically acceptable in a mammal, particularly a human.
  • FCA Freund's Complete adjuvant
  • FIA Freund's incomplete adjuvant
  • adjuvants include, but are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvant such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
  • Mineral salt adjuvants include but are not limited to: aluminum hydroxide, aluminum phosphate, calcium phosphate, zinc hydroxide and calcium hydroxide.
  • the adjuvant composition further comprises a lipid of fat emulsion comprising about 10% (by weight) vegetable oil and about 1-2% (by weight) phospholipids.
  • the adjuvant composition further optionally comprises an emulsion form having oily particles dispersed in a continuous aqueous phase, having an emulsion forming polyol in an amount of from about 0.2% (by weight) to about 49% (by weight), optionally a metabolizable oil in an emulsion- forming amount of up to 15% (by weight), and optionally a glycol ether-based surfactant in an emulsion-stabilizing amount of up to about 5% (by weight).
  • An intracellular delivery platform is provided herein which utilizes a genetically modified bacterium to deliver preventative or therapeutic anti-infective activity, immunomodulatory factors, or growth-promoting biomolecules directly to the mucosa of an animal in need thereof.
  • a novel delivery platform for delivering antigens, immunogens, antibodies, bioactive peptides, RNAs and other biotherapeutics and therapeutic biomolecules is provided.
  • bacteria such as Salmonella as a vector or means to deliver therapeutic biomolecules, such as immune, prophylactic or other applicable therapeutic biomolecules provides a number of applicable products and therapies targeting multiple disease conditions across a range of host species.
  • attenuated bacterial strains can be modified to deliver heterologous antigens, for example, as chromosomal or plasmid integrated genes, or a payload of eukaryotic antigen-expression plasmids (so-called DNA vaccines).
  • DNA vaccines eukaryotic antigen-expression plasmids
  • RNA-based vaccines both messenger RNA (mRNA) and self-amplifying replicons (SAM) are emerging as an increasingly promising alternative to traditional plasmid DNA for gene vaccination (DNA vaccines) (reviewed in Bloom K et al (2021) Gene Therapy 28:117-129; doi.org/ 10.1038/s41434-020-00204-y).
  • DNA vaccines have been shown to elicit antigen specific antibody and cellular immune responses against several viral pathogens with some clear advantages over DNA.
  • bacteria such as Salmonella, particularly attenuated bacteria such as Salmonella
  • therapeutic biomolecules such as immune, prophylactic or other applicable therapeutic biomolecules as part of a self-amplifying nucleic acid, particularly as self-amplifying mRNA (SAM) cargo
  • SAM self-amplifying mRNA
  • a novel vector and delivery platform is provided for delivering antigens, immunogens, antibodies, bioactive peptides, RNAs and other biotherapeutics and therapeutic biomolecules.
  • a novel vector and delivery platform is provided for delivering immunogens, antibodies and therapeutic biomolecules as vaccines, including prophylactic and therapeutic vaccines.
  • one or more biotherapeutic or therapeutic biomolecule is provided via the bacterial genomic nucleic acid, such as by means of integration, such as integration in the bacterial chromosome.
  • one or more biotherapeutic or therapeutic biomolecule is provided via a plasmid or vector which is retained by or otherwise replicated and expressed in the attenuated bacteria.
  • the plasmid or vector is retained via selection and selective growth of the bacteria retaining such plasmid or vector.
  • the plasmid or vector is capable of self-amplifying.
  • the term “self-amplifying” means capable of independent or self amplification, such as amplification independent of cellular or host proteins or factors, such as in the case of a self-amplifying nucleic acid, wherein a nucleic acid can amplify or make copies of itself independently.
  • the plasmid or vector is a self-amplifying nucleic acid, and is selected from self-amplifying DNA and self-amplifying RNA.
  • the self-amplifying DNA is an DNA-based sequence or vector or plasmid.
  • the self-amplifying RNA is an RNA-based sequence or vector or plasmid.
  • Positive-sense alphavirus genomes that have been commonly used for self-amplifying RNA vaccine design include the Venezuelan equine encephalitis virus (VEE), Sindbis virus (SINV), and Semliki forest virus (SFV), classical swine fever virus (CSFV), Sindbis virus (SINV), tick-borne encephalitis virus (TBEV).
  • VEE Venezuelan equine encephalitis virus
  • SINV Sindbis virus
  • SFV Semliki forest virus
  • CSFV classical swine fever virus
  • Sindbis virus SINV
  • tick-borne encephalitis virus TBEV
  • the alphavirus replicase genes encode an RNA-dependent RNA polymerase complex which amplifies synthetic transcripts in situ.
  • the antigenic or therapeutic sequence is expressed at high levels as a separate entity and further proteolytic processing of the immunogen is not required.
  • the intracellular delivery platform of the present disclosure includes a genetically modified bacterium having a self-amplifying nucleic acid.
  • the genetically modified bacterium includes bacterium from the Genus Salmonella.
  • the Salmonella bacterium includes Salmonella enterica. Salmonella enterica strains may be selected from S. Typhimurium, S. Enteriditis, S. Heidelberg, S. Gallinarum, S. Hadar, S. Agona, S. Kentucky, S. Typhi, S. Paratyphi, and S. Infantis.
  • the genetically modified bacterium includes Salmonella enterica, particularly Salmonella enterica serovar Typhimurium strain.
  • the genetically modified bacterium includes an attenuated bacterium.
  • attenuated means that the bacteria is reduced in causing disease symptoms in a host it is delivered to compared to a non-attenuated bacteria.
  • An attenuated bacterium may be an avirulent derivative of a pathogenic bacterium.
  • An attenuated bacterium includes wherein the bacterium is altered, for example by one or more mutation, so that the bacterium is avirulent, or incapable of causing a fulminant or significant infection in the host, and may also be incapable of survival in the host.
  • An attenuated bacterium includes wherein the bacterium is altered, for example by one or more mutation, so that the bacterium is avirulent, or incapable of causing a fulminant or significant infection in the host but that is capable of invoking or eliciting an immune response in a host.
  • Suitable attenuated bacteria can be any species or strain that is or can be sufficiently attenuated to allow for its non-pathological administration to humans and/or animals in live and/or dead form.
  • Attenuated bacterium/bacteria include an altered bacterium of a pathogenic organism comprising one or more inactivating mutation in a protein of the bacterium, such that the altered bacterium is effective in invoking an immune response in a host against the pathogenic organism.
  • Attenuated bacterium/bacteria include an altered pathogenic bacterium comprising one or more inactivating mutation in a protein of the bacterium, such that the altered bacterium is effective in protecting a host against subsequent pathogenic infection by an unaltered bacterium of the same or a related species or strain.
  • Attenuation can be accomplished by any method known in the art.
  • a bacterium may be attenuated by metabolic drift or by mutation of one or more genes.
  • attenuation is accomplished by genetic modification of a bacterium.
  • attenuation is accomplished by genetic modification of a gene of a bacterium, particularly a gene that is important for survival of the bacteria inside or outside the host.
  • the gene that is mutated provides an important aspect for the growth or survival of the bacterium. Mutation of the gene, including one or more genes, results in a bacterium that is altered, particularly one that is deficient, in one or more aspects required for growth or survival, particularly including in a bacterial host.
  • mutation includes any alteration in one or more nucleic acids in a genomic sequence, including one or more base changes, deletions, and/or insertions, that result in silent mutations, non-sense mutations, mis-sense mutations, or any such other mutations that result in reduced function of a gene or result in an inactive or otherwise non-functional protein encoded by a gene. Mutations include but are not limited to mutations that result in premature stop codons, aberrant splicing, altered or failed transcription, or altered or failed translation. A gene comprising a mutation can have more than one mutation. Mutations include deletion of a gene or a significant portion of a gene, particularly such that the gene’s protein is not produced or expressed and/or is inactive.
  • Mutations include insertions, such as wherein a foreign or heterologous sequence or nucleic acid is introduced into or otherwise inserted in the gene. Such insertion may block or eliminate translation to active or full length protein, or may result in a significantly altered and distinct protein that is not active as the wild type. An insertion may facilitate isolation, detection, selection of the gene mutant, such as by introduction or insertion of an antibiotic resistance gene or a detectable marker or protein.
  • the mutation including one or more mutation, is a non-natural mutation and is genetically engineered or recombinantly generated. In some embodiments, the mutation is genetically engineered or generated recombinantly in vitro.
  • the mutation is genetically engineered or generated recombinantly in a cell.
  • a mutation is generated whereby a gene, or a large or significant portion of a gene or protein encoding nucleic acid, is deleted.
  • one or more gene or a large or significant portion of a gene or protein encoding nucleic acid is deleted for example via recombination methods. Recombination methods for targeted deletion of genes are known and available to one skilled in the art.
  • Such methods include homologous recombination, such as via an introduced plasmid, phage or nucleic acid such as DNA or linear DNA fragment(s), recombination enzymes or recombinase enzyme mediated recombination, for example via recombinase recognition or target sequences, transposon mediated recombination and gene replacement.
  • deletion or inactivation mutations have been generated whereby one or more gene(s), or portions of one or more gene(s) so as to inactivate the gene(s) encoded protein, are deleted or inactivated in the genome of S. typhimurium bacteria.
  • Deletion mutants have thus been generated and utilized or have been utilized whereby deletions in each of the genes are constructed to provide new S. typhimurium mutant strains of bacteria. In some embodiments, these mutant bacteria are altered in growth. In some embodiments, these mutant bacteria are altered in survival, including in their stability in phagosomes or in bacteria –containing vacuoles, such as Salmonella-containing vacuoles (SCV).
  • SSV Salmonella-containing vacuoles
  • Methods to specifically inactivate one or more of the genes selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2 and asd are provided herein.
  • the bacterial pabA gene encodes an essential protein that converts glutamine to glutamate in the presence of stoichiometric amounts of pabB protein, which is encoded by the pabb gene. Mutant S. typhimurium bacteria which are ⁇ cya, ⁇ cyp, pabA mutants have been evaluated for intra-nasal immunogenicity in horses (Sheoran AS et al (2001) Vaccine 19(25-26):3591-3599).
  • the aroA gene encodes a protein enzyme involved in and required for aromatic amino acid synthesis.
  • the attenuated bacterium includes a Salmonella having a mutation in at least one of the following genes cya, crp, phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, and aroA.
  • the attenuated bacterium includes a Salmonella having a mutation in the cya gene and the crp gene.
  • the attenuated bacterium includes a Salmonella having at least one mutation selected from cya and crp, and mutation in at least one gene selected from cya, crp, phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, sspH2, paba, pabb, asd, and aroA.
  • the attenuated bacterium includes a Salmonella having at least one mutation selected from cya and crp, and a mutation in at least one gene selected from cya, crp, phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, sspH2 and mgtRBC.
  • the attenuated bacteria includes a Salmonella additionally having a mutation in at least one gene selected from paba, pabb, asd, and aroA.
  • the attenuated bacteria includes a Salmonella additionally having a mutation in at least one gene selected from paba, pabb, and aroA. In embodiments, the attenuated bacteria includes a Salmonella additionally having a mutation in asd. [000164] In some embodiments, the attenuated bacterium includes a Salmonella having a mutation in the genes for cya and crp, and a mutation in at least one gene selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, sspH2 and mgtRBC.
  • the attenuated bacteria includes a Salmonella additionally having a mutation in at least one gene selected from paba, pabb, asd, and aroA. In embodiments, the attenuated bacteria includes a Salmonella additionally having a mutation in at least one gene selected from paba, pabb and aroA. In embodiments, the attenuated bacteria includes a Salmonella additionally having a mutation in asd.
  • the attenuated bacterium includes a Salmonella having at least one mutation selected from cya and crp, and at least one mutation selected from cya, crp, phoPQ, ompR- envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, mgtRBC, paba, pabb, asd, and aroA.
  • the attenuated bacterium includes a Salmonella having at least one mutation selected from cya and crp, and at least one mutation selected from cya, crp, phoPQ, ompR- envZ, ssrAB, SPI2, sifA, sopF, feoABC, SPI 13 and sspH2.
  • the attenuated Salmonella bacterium includes a Salmonella having at least one mutation selected from cya and crp, and at least one mutation selected from cya, crp, phoPQ, ompR-envZ, sifA, and feoABC.
  • the attenuated Salmonella bacterium includes a Salmonella having at least one mutation selected from cya and crp, and at least one mutation selected from cya, crp, phoPQ, ompR-envZ, sifA, sopF and sspH2.
  • the attenuated bacterium includes a Salmonella having at least one mutation selected from cya and crp, and at least one mutation selected from cya, crp, phoPQ, ompR- envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, sspH2 and mgtRBC.
  • the attenuated bacteria includes a Salmonella additionally having a mutation in asd.
  • the attenuated bacteria includes a Salmonella additionally having a mutation in at least one gene selected from paba, pabb, asd, and aroA.
  • the attenuated bacteria includes a Salmonella additionally having a mutation in at least one gene selected from paba, pabb and aroA.
  • Salmonella bacteria are provided having a mutation in at least one of the following genes cya, crp, phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, sspH2 and mgtRBC.
  • Attenuated Salmonella bacteria having a mutation in the cya and crp genes and at least one of the following genes phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, sspH2 and mgtRBC.
  • Attenuated Salmonella bacteria are provided having a mutation in the cya and crp genes and at least one of the following genes phoPQ, ompR-envZ, sifA, sopF, feoABC, and ssPH2.
  • Attenuated Salmonella bacteria are provided having a mutation in the cya and crp genes and phoPQ. Attenuated Salmonella bacteria are provided having a mutation in the cya and crp genes and ompR-envZ. Attenuated Salmonella bacteria are provided having a mutation in the cya and crp genes and sifA. Attenuated Salmonella bacteria are provided having a mutation in the cya and crp genes and sopF. Attenuated Salmonella bacteria are provided having a mutation in the cya and crp genes and feoABC. Attenuated Salmonella bacteria are provided having a mutation in the cya and crp genes and ssPH2.
  • Attenuated Salmonella bacteria are provided having a mutation in at least one of the following genes phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, sspH2 and mgtRBC.
  • Attenuated Salmonella bacteria are provided having a mutation in at least one of the following genes phoPQ, ompR-envZ, sifA, sopF, feoABC, and ssPH2.
  • the attenuated Salmonella mutant(s) are auxotrophs, and require a particular additional nutrient which normal Salmonella strains do not.
  • the mutants exhibit growth defects in broth without added glucose. This auxotrophic property of these mutant provides a further attenuation characteristic and renders them more suitable candidates for vaccines or as a delivery vector, for example.
  • a vaccine or immunogenic composition comprising an attenuated Salmonella bacterium as disclosed herein.
  • a vaccine or immunogenic composition or immunogen comprising one or more attenuated Salmonella bacterium having at least one mutation selected from cya and crp, and at least one mutation selected from cya, crp, phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, sspH2 and mgtRBC is provided.
  • a vaccine or immunogenic composition or immunogen comprising one or more attenuated Salmonella bacterium having a mutation cya and crp, and at least one mutation selected from phoPQ, ompR-envZ, ssrAB, SPI2, sifA, sseJ, sopF, SPI 13, sitABCD, feoABC, sspH2 and mgtRBC is provided.
  • the gene mutation is a gene deletion mutation.
  • the gene mutation is a deletion generated by recombination, including wherein a substantive portion of the encoding region of the gene is deleted.
  • the attenuated bacterium is avirulent and immunogenic. In some embodiments, the attenuated bacterium is avirulent and serves to protect the host or administered animal against further infection by a non-attenuated bacterium or the same strain, related strains or the same species or genus. The attenuated bacterium may or may not elicit a humoral antibody response against the bacterium in the host or administered animal. Examples of attenuated bacterium include genetically modified bacterium disclosed in US Patent No.
  • US 5,389,368 the contents of which are incorporated by reference in their entirety.
  • attenuated bacterium examples include S. typhimurium strain X4062 and X4064, which have been deposited at the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110 USA.
  • ATCC American Type Culture Collection
  • U.S. Patent 5,389,368 describes an avirulent or attenuated Salmonella bacterium that contains an inactivating mutation in the gene encoding adenylate cyclase (cya) and in the gene encoding cyclic AMP receptor protein (cyp).
  • An exemplary such altered Salmonella bacterium corresponds to the attenuated Salmonella typhimurium bacteria strain component of the commercially available Megan ® Vac (AviPro ® ; Elanco).
  • Attenuation may be assessed or determined using any means and methods known in the art. For example, MeganVac bacteria was evaluated as a modified live vaccine to prevent wild type Salmonella typhimurium colonization of chickens with experimental challenge (McReynolds JL (2007) J Appl Poult Res 16:456-463). Such suitable assessments and systems can be utilized to evaluate alternative attenuated bacteria, including attenuated Salmonella, including any such attenuated Salmonella as provided herein.
  • the attenuated bacterium according to the present disclosure includes self-amplifying nucleic acid.
  • a self-amplifying nucleic acid is defined as a nucleic acid which is capable of independent or self amplification, such as amplification independent of cellular or host proteins or factors. The nucleic acid can amplify or make copies of itself independently.
  • Self-amplifying messenger RNA (denoted “SAM” herein) in its simplest form as utilized herein is an RNA based expression platform leveraging an engineered, non- disseminating viral genome.
  • the SAM platform provided herein is built on that of the alphavirus, e.g., Venezuelan equine encephalitis virus (VEEV), Semliki Forest virus (SFV), or Sindbis virus (SINV), classical swine fever virus (CSFV), Sindbis virus (SINV), tick-borne encephalitis virus (TBEV).
  • Alphavirus is a genus of RNA viruses, the sole genus in the Togaviridae family. The family of alphaviruses can infect humans, rodents, fish, birds, and larger mammals such as horses, as well as invertebrates (insects).
  • the alphaviruses have a genome of a single strand of positive-sense RNA.
  • the four non-structural protein (ns1-ns4) genes – necessary for transcription and replication of viral RNA - are encoded in the 5′ two-thirds of the genome, while the three structural proteins are translated from a subgenomic mRNA colinear with the 3′ one-third of the genome.
  • the three structural proteins are the core nucleocapsid protein C, and the envelope proteins P62 and E1, which associate as a heterodimer.
  • the genome encodes two polyproteins, the first consisting of the four non-structural units, in order from the N terminal to the C terminal - nsP1, nsP2, nsP3, and nsP4, the second being is a structural polyprotein composed of five expression units: from the N terminal to the C terminal - Capsid, E3, E2, 6K and E1.
  • Alphaviruses include Eastern equine encephalitis virus (seven antigenic types), Middelburg virus, Ndumu virus, Semliki Forest virus, Venezuelan equine encephalitis virus, Western equine encephalitis and Sindbis virus.
  • the "replication engine” of alphavirus is responsible for amplifying the viral genome through a negative strand RNA intermediate.
  • the second alphavirus functional unit, encoding the structural proteins can be replaced with any transgene of choice or heterologous gene of interest.
  • SAM RNA will create many copies of itself, acting as the template for translation of the novel transgene or heterologous gene.
  • the presently provided SAM system will produce significantly more transgene or gene of interest and protein or peptide encoded therefrom (e.g., vaccine antigen or biotherapeutic) because of the self- amplifying nature.
  • the SAM is no longer infectious or virulent.
  • the SAM can be design and adjusted so as to provide different levels or extent of immunogenicity to accommodate both vaccine (high immunogenicity) and bio-therapeutic (low immunogenicity) applications.
  • the second alphavirus functional unit, encoding the structural proteins is replaced with one or more gene of interest, heterologous gene, or transgene.
  • the SAM may include one or more gene of interest.
  • the gene of interest may particularly be a biotherapeutic or therapeutic biomolecule.
  • the SAM may include a chimeric protein or fusion protein.
  • a chimeric protein or fusion protein includes wherein a first heterologous protein of interest is combined with another distinct protein or peptide of interest.
  • a chimeric protein or fusion protein includes wherein a first heterologous protein of interest is combined with a targeting protein or targeting sequence which may direct the first heterologous protein to a particular cell type, a particular cell receptor, or a tissue or region of the body of an animal for instance.
  • a chimeric protein or fusion protein includes wherein a first heterologous protein of interest is combined with a targeting protein or targeting sequence which may direct the first heterologous protein outside of the cell of expression, such as to be expressed or located systemically in an animal, or to the blood local tissues in the animal.
  • a chimeric protein includes wherein a first heterologous protein is combined with a label, tag or enzyme.
  • a tag or label or enzyme may be a functional molecule.
  • a tag or label may be an epitope.
  • a tag or label may be a detectable molecule, protein or other entity.
  • a tag or label may be a fluorescent molecule, a radioactive molecule, etc. Suitable fluorescent molecules are known and available in the art.
  • a fluorescent molecule may be a green fluorescent protein (GFP) for example.
  • GFP green fluorescent protein
  • the self amplifying nucleic acid encode for molecules required for its self-amplification and a therapeutic or biologically active molecule
  • the self amplifying nucleic acid particularly may encode for at least one of nsP1, nsP2, nsP3, and nsP4, and a therapeutic or biologically active molecule, which may be selected from a protein, such as a polypeptide, enzyme, antigen, and a nucleic acid, such as RNA or DNA.
  • the self amplifying nucleic acid encodes for the replication proteins nsP1, nsP2, nsP3, and nsP4, and also one or more therapeutic or biologically active molecule, which may be selected from a protein, such as a polypeptide, enzyme, antigen, and a nucleic acid, such as RNA or DNA.
  • the nucleic acid that encodes for the therapeutic or biologically active molecule may be under the control of a heterologous promoter.
  • the nucleic acid that encodes for the therapeutic or biologically active molecule may be under the control of a subgenomic promoter.
  • a subgenomic promoter includes a transcriptional promoter, such as the alphavirus subgenomic promoter (also referred to as the 26S promoter or the viral junction region promoter).
  • a transcriptional promoter such as the alphavirus subgenomic promoter (also referred to as the 26S promoter or the viral junction region promoter).
  • M cells mucosal cells
  • dendritic cells and macrophages While in the dendritic cell or macrophage, the Salmonella is lysed, thereby releasing the self-amplifying nucleic acid.
  • the therapeutic or biologically active molecule may be any molecule, including a polypeptide or nucleic acid, having a useful or desired activity.
  • a therapeutic biomolecule includes a biomolecule having a therapeutic effect. Examples of therapeutic biomolecules include polypeptide (eg, GLP-1), enzyme, antibody, a ribonucleic acid (RNA), and antigen.
  • Antibody includes antibody fragments, such as VHH.
  • RNA includes inactivating RNA, such as shRNA and siRNA.
  • Antigen includes a biomolecule that stimulates an immune response. Examples of antigens include a peptide, polypeptide, protein, nucleic acid molecule, and carbohydrate molecule.
  • the molecule may be selected from an antibody, a ribonucleic acid (RNA), a peptide or protein, and an antigen.
  • RNA ribonucleic acid
  • antigens particularly Eimeria antigens
  • the molecule may be selected from an antibody, a ribonucleic acid (RNA), a peptide or protein, and an antigen.
  • Antibodies in accordance with the present disclosure include an immunoglobulin and particularly any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. CDR grafted antibodies are also contemplated by this term.
  • An "antibody” is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope. The term encompasses polyclonal, monoclonal, and chimeric antibodies.
  • antibody(ies) includes a wild type immunoglobulin (Ig) molecule, generally comprising four full length polypeptide chains, two heavy (H) chains and two light (L) chains, or an equivalent Ig homologue thereof (e.g., a camelid nanobody, which comprises only a heavy chain); including full length functional mutants, variants, or derivatives thereof, which retain the essential epitope binding features of an Ig molecule, and including dual specific, bispecific, multispecific, and dual variable domain antibodies. Also included within the meaning of the term “antibody” are any “antibody fragment”.
  • an “antibody fragment” refers to a molecule comprising at least one polypeptide chain that is not full length, including (i) a Fab fragment, which is a monovalent fragment consisting of the variable light (VL), variable heavy (VH), constant light (CL) and constant heavy 1 (CH1) domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a heavy chain portion of an Fab (Fd) fragment, which consists of the VH and CH1 domains; (iv) a variable fragment (Fv), which consists of the VL and VH domains of a single arm of an antibody, (v) a domain antibody (dAb) fragment, which comprises a single variable domain (Ward, E.S.
  • a Fab fragment which is a monovalent fragment consisting of the variable light (VL), variable heavy (VH), constant light (CL) and constant heavy 1 (CH
  • a minibody which is a bivalent molecule comprised of scFv fused to constant immunoglobulin domains, CH3 or CH4, wherein the constant CH3 or CH4 domains serve as dimerization domains (Olafsen T et al (2004) Prot Eng Des Sel 17(4):315-323; Hollinger P and Hudson PJ (2005) Nature Biotech 23(9):1126-1136); and (xiii) other non-full length portions of heavy and/or light chains, or mutants, variants, or derivatives thereof, alone or in any combination. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are also included.
  • an "antibody combining site" is that structural portion of an antibody molecule comprised of light chain or heavy and light chain variable and hypervariable regions that specifically binds antigen.
  • Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope, including those portions known in the art as Fab, Fab', F(ab')2 and F(v).
  • Antibodies may also be bispecific, wherein one binding domain of the antibody has a first binding specificity, and the other binding domain has a different specificity, e.g. to recruit an effector function or the like.
  • the other binding domain may be an antibody that recognizes or targets a particular cell type or to recognize particular cell receptors and/or modulate cells in a particular fashion, as for instance an immune modulator (e.g., interleukin(s)), a growth modulator or cytokine or a toxin (e.g., ricin) or anti-mitotic or apoptotic agent or factor.
  • an immune modulator e.g., interleukin(s)
  • cytokine e.g., ricin
  • anti-mitotic or apoptotic agent or factor e.g., interleukin(s)
  • the term “antigen binding domain” describes the part of an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may bind to a particular part of the antigen only, which part is termed an epitope.
  • An antigen binding domain may be provided by one or more antibody variable domains.
  • An antigen binding domain may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), or may only comprise an antibody heavy chain variable region (VH).
  • Immunoconjugates or antibody fusion proteins are also contemplated, wherein the antibodies, antibody molecules, or fragments thereof, applicable herein are conjugated or attached to other molecules or agents. Such immunoconjugates or antibody fusion proteins may further include, but are not limited to such antibodies, molecules, or fragments conjugated to a chemical ablation agent, toxin, immunomodulator, cytokine, cytotoxic agent, chemotherapeutic agent, antimicrobial agent or peptide, cell wall and/or cell membrane disrupter, or drug.
  • Single domain antibodies are included as a particular embodiment of the therapeutic or biologically active molecules delivered in accordance with the intracellular delivery platform provided herein and expressed by the self amplifying nucleic acid.
  • Single domain antibodies are initially isolated from camelid animals and have been designated interchangeably as camelid antibodies, nanobodies or VHH.
  • a VHH antibody corresponds to the variable region of an antibody heavy chain and has a very small size of around 15 kDa - hence the name "nanobody”.
  • the advantage of these antibody-derived molecules is their small size which can enable their binding to hidden epitopes not accessible to whole antibodies.
  • a small molecular weight also means an efficient penetration and fast clearance.
  • Nanobodies are small, low molecular weight, single-domain, heavy-chain only antibody found in camelids. Owing to its smaller size, genes of these proteins are easy to clone inside a plasmid. Therefore, by using molecular cloning techniques, nanobodies against various antigens can be presented in the systemic circulation.
  • the present embodiments, methods and intracellular delivery platform has been utilized to deliver and express antibody fragments, particularly VHH or nanobodies.
  • RNA interference is an evolutionary regulatory mechanism of cells that uses ⁇ 21-25 long siRNA transcripts to effectively control the expression of desired genes by inhibiting the expression of mRNA transcripts through degrading or binding sequence specifically, thus hindering translation into proteins.
  • the silencing mechanism occurs at the levels of transcription, post-transcription, and translation. RNAi can also cause augmentation of gene expression due to direct effects on the translation.
  • RNAi small ribonucleic acid
  • miRNA micro-RNA
  • siRNA small interfering RNA
  • shRNA small hairpin RNA
  • RNAi is a specific, potent, and highly successful approach for loss-of-function studies in virtually all eukaryotic organisms. Besides being a method of therapeutic target identification of drug discovery, knockdown of target genes with RNAi reagents also can be used to elucidate biochemical and cell signaling pathways.
  • a short hairpin RNA or small hairpin RNA is an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi).
  • RNA interference is a biological mechanism in which small RNA molecules bind to specific mRNA molecules and inhibit translation.
  • shRNA and siRNA against Porcine Reproductive and Respiratory Syndrome (PRRS) viral surface proteins docking CD163/SRCR5 host receptor have been designed and cloned into the SAM vector. Resulting plasmids are transformed into Salmonella Typhimurium.
  • Antigens are a substance, such as a protein or peptide, which induces an immune response, especially the production of antibodies.
  • an antigen is a molecule or molecular structure, such as may be present on the outside of a pathogen, that can be bound by an antigen-specific antibody or B-cell antigen receptor. The presence of antigens in the body normally triggers an immune response.
  • vaccines The basis of vaccines is the presentation of one or more antigen from an infectious agent to an animal or host, such that the animal or host has an immune response and raises antibodies against the infectious agent. This immune response and these raised antibodies serve to protect the host or animal from further infection, disease or illness by the infectious agent.
  • vaccines provided and contemplated herein are capable of and utilized to generate mucosal, systemic and cellular immunity against one or more pathogen(s).
  • An antigen may include all or a portion of a protein.
  • an antigen may be an antigenic portion or fragment of a full length protein.
  • An antigen may be a non-natural fragment of a protein.
  • the delivery platform may be utilized to express one or more antigen for a particular pathogen.
  • Avian coccidosis is a common poultry disease caused by Eimeria. Control of coccidosis has been approached by medicating feed with anticocciddial drugs or administering vaccines containing low doses of virulent or attenuated Eimeria oocysts. Problems of drug resistance and nonuniform administration of these Eimeria resulting in variable immunity prompt efforts to develop improved and recombinant Eimeria vaccines and other approaches to stimulate immunity and address cocciosis disease.
  • Eimeria is a genus of parasites that includes various species capable of causing the disease coccidiosis in animals such as cattle, poultry, dogs (especially puppies), cats (especially kittens), and smaller ruminants including sheep and goats. Species of this genus infect a wide variety of hosts. The most prevalent species of Eimeria that cause coccidiosis in cattle are E. bovis, E. zuernii, and E. auburnensis. [000201] Delivery of an antigen capable of generating an immune response via the live intraceullular delivery platform and using exemplary SAM vectors has been demonstrated herein.
  • Coccidial vaccine Salmonella Typhimurium was modified to deliver cross-protective antigens covering Eimeria tenella, E. maxima and E. acervulina as a part of SAM payload.
  • Eimeria tenella elongation factor -1 ⁇ SEQ ID NO: 155
  • EtAMA1 SEQ ID NO: 157
  • Eimeria tenella 5401 SEQ ID NO: 173
  • Eimeria acervulina lactate dehydrogenase antigen gene SEQ ID NO: 174
  • Eimeria maxima surface antigen gene SEQ ID NO: 156
  • Glyceraldehyde 3-phosphate Dehydrogenase (GAPDH) SEQ ID NO: 153
  • Eimeria common antigen 14-3-3 SEQ ID NO: 154) antigens are delivered and expressed in an applicable system.
  • Peptides or Proteins There are various peptides or proteins which act independently as therapeutic biomolecules. Among these are anti-infective or anti-bacterial peptides which can serve to block or treat infection by infectious agents or bacteria. [000203] A wide range of antimicrobial peptides is secreted in plants and animals to challenge attack by foreign viruses, bacteria or fungi (Boman, H. G. (2003) J. Intern. Med.254 (3):197-215). These form part of the innate immune response to infection, which is short term and fast acting relative to humoral immunity. These peptides are heterogeneous in length, sequence and structure, but most are small, cationic and amphipathic (Zasloff, M.
  • antimicrobial peptides are listed at an antimicrobial database (aps.unmc.edu/AP/main.php; Wang Z and Wang G (2004) NAR 32:D590-D592) and the content and disclosure of this site is incorporated herein by reference in its entirety. While the external cell wall may be the initial target, several lines of evidence suggest that antimicrobial peptides act by lysing bacterial membranes. Cells become permeable following exposure to peptides, and their membrane potential is correspondingly reduced.
  • a cell-wall degrading enzyme is an enzyme which degrades components of the cell wall, including peptidoglycans, such as murein and pseudomurein, chitin, and teichoic acid.
  • Cell-wall degrading enzymes can include, but are not limited to amidases, muramidases, endopeptidases, glucosaminidases.
  • Bacteriophage lysins are cell wall degrading anti-bacterial enzymes encoded by phage in bacteria. Lysins are peptidoglycan hydrolases that break bonds in the bacterial wall, rapidly hydrolyzing covalent bonds essential for peptidoglycan integrity, causing bacterial lysis and concomitant progeny phage release. Bacteriophage lytic enzymes have been established as useful in the assessment and specific treatment of various types of infection in subjects through various routes of administration. Phage associated lytic enzymes have been identified and cloned from various bacteriophages, each shown to be effective in killing specific bacterial strains. [000205] Streptococcus suis bacteria infects Streptococcus suis infects swine (pigs).
  • PlySs2 is a S.suis phage lytic enzyme capable of killing Streptococcus bacteria including S. suis in vitro and in vivo in animal models (Gilmer DB et al (2013) Antimicrob Agents Chemother 57(6):2743-2750; Gilmer DB et al (2017) PLoSONE 12(1):e0169180).
  • PlySs2 has both broad and high killing activity against multiple serotypes and strains of S. suis.
  • Strep suis (Swine)- Salmonella Typhimurium is modified to deliver Streptococcus suis specific Plyss2 lytic enzyme as a part of SAM payload assembly (systemic bio-pharmaceutical delivery).
  • compositions of attenuated bacteria having a self-amplifying nucleic acid are provided herein.
  • the self-amplifying nucleic acid encodes and is capable of facilitating expression of a therapeutic biomolecule.
  • the compositions include pharmaceutical compositions, immunogenic compositions.
  • the compositions may further include one or more component or additive.
  • the one or more component or additive may be a component or additive to facilitate administration, for example by way of a stabilizer or vehicle, or by way of an additive to enable administration to an animal such as by any suitable administrative means, including in aerosol or spray form, in water, in feed or in an injectable form. Administration to an animal may be by any known or standard technique.
  • compositions may include a pharmaceutical carrier in which the bacterium or any such other aspects or components of the intracellular delivery platform is suspended or dissolved.
  • carrier(s) may be any solvent or solid or encapsulated in a material that is non-toxic to the inoculated animal and compatible with the carrier organism or gene product such as the gene of interest or therapeutic biomolecule.
  • Suitable pharmaceutical carriers include liquid carriers, such as normal saline and other non-toxic salts at or near physiological concentrations, and solid carriers, such as talc or sucrose and which can also be incorporated into feed for farm animals.
  • Adjuvants may be added to enhance the antigenicity if desired.
  • the composition When used for administering via the bronchial tubes, the composition is preferably presented in the form of an aerosol.
  • a dye may be added to the compositions hereof, including to facilitate checking or confirming whether an animal has ingested or breathed in the composition.
  • the compositions disclosed herein can be formulated as animal feed, feed additive, food ingredient, vaccine additive or ingredient, water additive, water-mixed additive, consumable solution, consumable spray additive, consumable solid, consumable gel, injection, or combinations thereof.
  • the composition includes water.
  • administration When administering to animals, including farm animals, administration may include orally or by injection. Oral administration can include by bolus, tablet, or paste, or as a powder or solution in feed or drinking water.
  • the method of administration will often depend on the species being treated, the numbers needing treatment, and other factors such as the handling facilities available and the risk of stress for the animal.
  • the dosages required will vary with the gene product or therapeutic biomolecule and need be an amount sufficient to induce an immune response or to effect a biological change or response expected or desired. Routine experimentation will establish the required amount. Increasing amounts or multiple dosages may be implemented and used as needed.
  • the embodiments and aspects herein have wide applicability to the development of effective vaccines against bacterial, fungal, parasite or viral disease agents where local immunity is important and might be a first line of defense. Such vaccines may be applicable to hatchery or field vaccine programs, particularly in farm and feed animals. Viral vaccines can be produced against either DNA or RNA viruses.
  • Vaccines to protect against infection by pathogenic fungi, protozoa and parasites are also contemplated. Both therapeutic vaccines, such as wherein an antibody or portion thereof is administered and expressed via the delivery platform, for example to an animal with a disease or infection, and prophylactic vaccines, wherein a protein or antigen is administered and expressed via the delivery platform and serves to stimulate immunity in an animal are provided and contemplated herein.
  • therapeutic vaccines such as wherein an antibody or portion thereof is administered and expressed via the delivery platform, for example to an animal with a disease or infection
  • prophylactic vaccines wherein a protein or antigen is administered and expressed via the delivery platform and serves to stimulate immunity in an animal are provided and contemplated herein.
  • the following experimental examples are illustrative of a intracellular delivery platform comprising an attenuated bacterium, for example without limitation an attenuated Salmonella bacterium.
  • the attenuated bacterium When administered to a non-human animal, the attenuated bacterium is able to enter cells of the host animal, for example without limitation macrophages, dendritic cells, and M cells. Once inside a host cell, the attenuated bacterium releases a self-amplifying nucleic acid encoding a therapeutic biomolecule.
  • the therapeutic biomolecule has a desired effect on the animal, for example without limitation treating, ameliorating, or preventing a disease or disorder, or eliciting protective immunity against a pathogenic or parasitic organism.
  • Salmonella as a bacterial delivery platform offers significant biological advantages. Salmonella strains can infect multiple host species including humans and also commercially important animals in the food industry including poultry, cattle and swine. Specifically, Salmonella targets M cells overlying gut-associated lymphoid tissue (inductive sites for immune responses), are readily internalized by dendritic cells and macrophages; stimulate broad immune responses including serum antibodies, secretory IgA intestinal antibodies and an array of cell- mediated immune (CMI) responses, including cytotoxic lymphocytes (CTL) [1, 2, 3].
  • CLI cell- mediated immune
  • Attenuated bacterial vectors have been modified to deliver heterologous antigens, for example, as chromosomal or plasmid integrated genes or a payload of eukaryotic antigen expression plasmids (DNA vaccines) [1, 2, 3].
  • An attenuated Salmonella bacteria has been utilized in initial studies to provide an initial platform for delivery and to demonstrate the applicability of the present approach.
  • the commercial Salmonella vaccine strain from the AVIPRO MEGAN VAC1 vaccine product Megan ® Vac (AviPro ® ; Elanco), noted herein as “MEGAN VAC” or “MeganVac” is chosen for the live intracellular delivery platform studies provided herein.
  • the MEGAN VAC strain is defective in the cyclic AMP- cAMP receptor protein (CRP) global regulatory system. Because of the ⁇ cya and ⁇ crp deletions, this strain is avirulent but remains immunogenic.
  • CPP cyclic AMP- cAMP receptor protein
  • genomic DNA is isolated from overnight culture and whole genome sequencing performed using PACBIO sequencing.
  • Antibiotic susceptibility of this strain is analyzed by serial dilution method in a 96-well plate against selected antibiotics. Result of the antibiotic sensitivity test is presented in Table 1. Table 1. Antibiotic sensitivity of the MEGAN VAC Salmonella strain.
  • EXAMPLE 2 SELF AMPLIFYING RNA (SAM) [000217]
  • An alphavirus (family Togaviridae) is selected as a base for constructing a self-amplifying nucleic acid.
  • the alphavirus genome is a positive sense, single stranded RNA with the ability to self- replicate in the cytoplasm of an infected cell and produce large amounts of proteins.
  • Examples of an alphavirus are Chikungunya virus, Sindbis virus, Semliki Forest virus, the Ross River virus, and the western, eastern and Venezuelan equine encephalitis viruses.
  • the alphavirus RNA genome encodes four non-structural proteins (nsP1, nsP2, nsP3, and nsP4) in the 5’ region and viral structural protein genes at the 3’ end.
  • the structural protein genes are under the control of a single subgenomic promoter.
  • the structural genes may be replaced by one or more foreign gene of interest, particularly a gene encoding a therapeutic biomolecule, to achieve intracellular expression, particularly significant or high intracellular expression, and thereby can provide systemic expression or delivery of a therapeutic biomolecule.
  • the self-amplifying mRNA uses the host cell translation machinery to produce the functional viral genome replication proteins nsP1-nsP4, which provide the four functional components of RNA-dependent RNA polymerase. These proteins then form a functional replication factory and use the positive sense genomic RNA to transcribe the negative sense RNA.
  • the negative sense strand serves as a template to generate two types of RNA: a full- length genomic RNA and multiple copies of subgenomic messenger RNAs. This leads to very high level of expression of proteins via the self-amplifying mRNA, expressing structural proteins or foreign proteins or therapeutic biomolecules via the gene(s) of interest while generating fresh pool of positive sense genomic RNA.
  • FIGURE 1 Construction of an exemplary self-amplifying mRNA (SAM), denoted A69, is shown in FIGURE 1.
  • Plasmid A69 carries virus nonstructural RNA polymerase component proteins (nsPs), particularly alphavirus nsP1-4.
  • nsPs virus nonstructural RNA polymerase component proteins
  • CMV cytomegalovirus
  • any suitable high level expression promoter may be utilized.
  • Expression of a foreign gene or gene of interest is driven by the virus subgenomic promoter.
  • the foreign gene (denoted as gene of interest) is green fluorescence protein (GFP).
  • GFP green fluorescence protein
  • the exemplary plasmid A69 sequence is provided in SEQ ID NO: 126.
  • the encoding sequence of GFP (SEQ ID NO: 129) is as follows: [000223] Sequence of the subgenomic promoter (SEQ ID NO: 130) is: CTCTCTACGGCTAACCTGAATGGA [000224] Exemplary encoding plasmid nucleic acid sequences of the alphavirus ns1, ns2, ns3 and ns4 (from Venezuelan equine encephalitis virus (VEEV), are as follows:
  • Plasmid A69 is used to demonstrate the function of the live intracellular delivery platform. Plasmid was first transformed inside Salmonella typhimurium bacteria using standard methods, so as to confirm that the plasmid is stable and propogated inside Salmonella. Then, pA69 was electroporated into cells of the chicken macrophage cell line HD11. Green fluorescence is observed in transfected cells, which confirms functional expression of the GFP gene. [000226] To test the delivery of A69 using Salmonella, pA69 is transformed into an attenuated Salmonella, particularly the MaganVac having ⁇ cya and ⁇ crp deletions. HD11 cells are infected at a multiplicity of infection (MOI) of 100 with Salmonella carrying plasmid A69.
  • MOI multiplicity of infection
  • Phagocytes include monocytes and macrophages, granulocytes, and dendritic cells and these cells capable of engulfing and absorbing bacteria and other small cells and particles. Phagocytes kill bacteria by engulfing them and forming phagosomes. Once internalized, phagosomes fuse with endosomal vacuoles to form microbicidal phagolysosomes.
  • Salmonella bacteria produce a variety of phagosome-induced (or macrophage-induced) proteins as survival factors.
  • T3SS2 type 3 secretion system-2
  • Salmonella Typhimurium forms a needle like apparatus that injects survival factors into early phagosomes and prevents phagosome fusion with an endosome.
  • a direct result of such survival factors or effector proteins is that Salmonella bacteria can reside and proliferate inside the specialized Salmonella-containing vacuoles (SCV).
  • SCV Salmonella-containing vacuoles
  • Inactivation mutation or deletion of these genes singly or in combination, thus deletion or inactivation of one or more of these genes, to generate a mutant Salmonella bacteria should give rise to different and altered levels of virulence.
  • the goal is to create deletions to generate Salmonella bacteria which are susceptible to the environment of a SCV, thus facilitating lysis of the bacteria, killing it.
  • the payload or self- amplifying RNA construct will be released into the host cytosol and the foreign/heterologous gene will be expressed within the host cell.
  • a two-step homologous recombination method is performed.
  • a linear DNA fragment containing homologous regions of the target gene and a chloramphenicol acyl transferase cat cassette flanked by two FRT cassettes is constructed (Schlake, T. and J. Bode, Use of mutated FLP recognition target (FRT) sites for the exchange of expression cassettes at defined chromosomal loci. Biochemistry, 1994.33(43): p.12746-51).
  • the construct is electroporated inside a Salmonella bacterium containing the ⁇ -Red recombinase plasmid pSIJ8 (Jensen et al Sci Rep.2015 Dec 8;5:17874. doi: 10.1038/srep17874).
  • Recombination is performed by FLP recombinase, under the control of a rhamnose promoter on the pSIJ8 plasmid. Following recombination, the target gene is replaced by cat. The resulting mutant bacteria strain is chloramphenicol sensitive and the survival factor gene deletion is confirmed by PCR. After recombination, the bacteria is cured of the plasmid and the deletion is retained in the bacterial genomic DNA. A schematic representation of this process is provided in FIGURE 3A. [000230] The target genome is based on that of parent strain Salmonella enterica Serovar Typhimurium UK1 atrsin (ATCC 68169).
  • Typhimurium crp-cya mutant is the first and the most commonly used live attenuated vaccine in the United States (Curtiss R, 3rd, Hassan JO. Vet Immunol Immunopathol. l 996;54(1-4):365-72; Curtiss, R., 3rd and S. M. Kelly (1987). Infection and immunity 55(12): 3035-3043; Hassan JO, Curtiss R, 3rd. Res Microbiol.1990;141(7-8):839-50; Hassan JO, Curtiss R, 3rd.
  • PhoPQ is a histidine-kinase protein switch that senses the internal environment of SCV and regulates several Salmonella genes. Increased expression of PhoPQ results in increased uptake of Ca 2+ and Mg 2+ ions.
  • a genomic nucleic acid sequence for PhoPQ is provided as SEQ ID NO: 1.
  • PCR primer pairs are provided for the upstream (SEQ ID NOs: 2 and 3) and downstream (SEQ ID NOs: 4 and 5) regions of the PhoPQ locus. Successful deletion of the targeted chromosomal region is assessed by the primer pair provided as SEQ ID NOs: 6 and 7).
  • Other primer pairs useful in making this construct are directed to the pUC19 vector (SEQ ID NOs: 8 and 9), the first half of the cat insertion (SEQ ID NOs: 10 and 11), the second half of the cat insertion (SEQ ID NOs: 12 and 13), and the FRT cassette (SEQ ID NOs: 14 and 15).
  • SPI2 genes within a pathogenicity island result in the needle-like apparatus of T3SS2.
  • Factors encoded by this pathogenicity island are responsible for injecting other effector proteins into the cytosol of the host cell.
  • a genomic nucleic acid sequence for SPI2 is provided as SEQ ID NO: 16.
  • PCR primer pairs are provided for the upstream (SEQ ID NOs: 17 and 18) and downstream (SEQ ID NOs: 19 and 20) regions of the SPI2 locus. Successful deletion of the targeted chromosomal region is assessed by the primer pair provided as SEQ ID NOs: 21 and 22.
  • OmpR-envZ is a two-component master regulator which directs expression of the ssrAB complex, among other factors. This master regulator senses the low pH condition inside phago- lysosomes and protects Salmonella bacteria from this acidic environment.
  • a genomic nucleic acid sequence for OmpR-envZ is provided as SEQ ID NO: 27.
  • PCR primer pairs are provided for the upstream (SEQ ID NOs: 28 and 29) and downstream (SEQ ID NOs: 30 and 31) regions of the OmpR- envZ locus. Successful deletion of the targeted chromosomal region is assessed by the primer pair SEQ ID NOs: 32 and 33.
  • Other primers useful in making this construct are directed to the pUC19 vector (SEQ ID NOs: 8 and 9), the first half of the cat insertion (SEQ ID NOs: 34 and 11), the second half of the cat insertion (SEQ ID NOs: 12 and 35), and the FRT cassette (SEQ ID NOs: 36 and 37).
  • SsrAB is another two-component regulon that directs expression of SPI2 genes.
  • a genomic nucleic acid sequence for ssrAB is provided as SEQ ID NO: 38.
  • PCR primer pairs are provided for the upstream (SEQ ID NOs: 39 and 40) and downstream (SEQ ID NOs: 41 and 42) regions of the ssrAB locus. Successful deletion of the targeted chromosomal region is assessed by the primer pair provided as SEQ ID NOs: 43 and 44.
  • Other primers useful in making this construct are directed to the pUC19 vector (SEQ ID NOs: 8 and 9), the first half of the cat insertion (SEQ ID NOs: 45 and 11), the second half of the cat insertion (SEQ ID NOs: 12 and 46), and the FRT cassette (SEQ ID NOs: 47 and 48).
  • SifA is a T3SS2 effector which is required for the formation of Salmonella induced filamentation (SIF). SifA plays a key role in blocking phagosome maturation, allowing Salmonella to survive and replicate inside phagocytes. It has been previously demonstrated that sifA mutants are defective for survival and replication within macrophages and attenuated for virulence in mice and that sifA mutants lose vacuolar membranes at late stages of endosome maturation releasing Salmonella into the cytosol [7, 8].
  • a genomic nucleic acid sequence for SifA is provided as SEQ ID NO: 49.
  • PCR primer pairs are provided for the upstream (SEQ ID NOs: 50 and 51) and downstream (SEQ ID NOs: 52 and 53) regions of the SifA locus. Successful deletion of the targeted chromosomal region is assessed by the primer pair provided as SEQ ID NOs: 54 and 55.
  • Other primers useful in making this construct are directed to the pUC19 vector (SEQ ID NOs: 8 and 9), the first half of the cat insertion (SEQ ID NOs: 56 and 11), the second half of the cat insertion (SEQ ID NOs: 12 and 57), and the FRT cassette (SEQ ID NOs: 58 and 59).
  • SseJ is a T3SS2 effector protein which, in conjunction with another effector molecule sseL, is required to maintain the SCV integrity.
  • a genomic nucleic acid sequence for sseJ is provided as SEQ ID NO: 60.
  • PCR primer pairs are provided for the upstream (SEQ ID NOs: 61 and 62) and downstream (SEQ ID NOs: 63 and 64) regions of the sseJ locus. Successful deletion of the targeted chromosomal region is assessed by the primer pair provided as SEQ ID NOs: 65 and 66.
  • SopF is a T3SS1 effector molecule that is required for SCV membrane integrity.
  • a genomic nucleic acid sequence for sopF is provided as SEQ ID NO: 71.
  • PCR primer pairs are provided for the upstream (SEQ ID NOs: 72 and 73) and downstream (SEQ ID NOs: 74 and 75) regions of the sseJ locus.
  • SPI13 is a pathogenicity island required to protect Salmonella from methylglyoxal detoxification.
  • a genomic nucleic acid sequence for SPI13 is provided as SEQ ID NO: 82.
  • PCR primer pairs are provided for the upstream (SEQ ID NOs: 83 and 84) and downstream (SEQ ID NOs: 85 and 86) regions of the SPI13 locus. Successful deletion of the targeted chromosomal region is assessed by the primer pair provided as SEQ ID NOs: 87 and 88.
  • Other primers useful in making this construct are directed to the pUC19 vector (SEQ ID NOs: 8 and 9), the first half of the cat insertion (SEQ ID NOs: 89 and 11), the second half of the cat insertion (SEQ ID NOs: 12 and 90), and the FRT cassette (SEQ ID NOs: 91 and 92).
  • SitABCD genes are located in the SPI1 pathogenicity island, and these are highly upregulated once Salmonella enters a phagocyte. This complex is required for protection against Mn 2+ starvation inside the SCV.
  • a genomic nucleic acid sequence for sitABCD is provided as SEQ ID NO: 93.
  • PCR primer pairs are provided for the upstream (SEQ ID NOs: 94 and 95) and downstream (SEQ ID NOs: 96 and 97) regions of the sitABCD locus. Successful deletion of the targeted chromosomal region is assessed by the primer pair provided as SEQ ID NOs: 98 and 99.
  • FeoABC is an operon which is highly up-regulated inside a phagocyte and is required for Fe 2+ uptake as Salmonella replicate inside a phagocyte.
  • a genomic nucleic acid sequence for feoABC is provided as SEQ ID NO: 104.
  • PCR primer pairs are provided for the upstream (SEQ ID NOs: 105 and 106) and downstream (SEQ ID NOs: 107 and 108) regions of the feoABC locus. Successful deletion of the targeted chromosomal region is assessed by the primer pair provided as SEQ ID NOs: 109 and 110.
  • Other primers useful in making this construct are directed to the pUC19 vector (SEQ ID NOs: 8 and 9), the first half of the cat insertion (SEQ ID NOs: 111 and 11), the second half of the cat insertion (SEQ ID NOs: 12 and 112), and the FRT cassette (SEQ ID NOs: 113 and 114).
  • MgtRBC similar to feoABC, is an operon which is also highly upregulated inside phagocytes. MgtRBC is required for Mg 2+ uptake.
  • a genomic nucleic acid sequence for mgtRBC is provided as SEQ ID NO: 115.
  • PCR primer pairs are provided for the upstream (SEQ ID NOs: 116 and 117) and downstream (SEQ ID NOs: 118 and 119) regions of the mgtRBC locus. Successful deletion of the targeted chromosomal region is assessed by the primer pair provided as SEQ ID NOs: 120 and 121.
  • sspH2 is a Salmonella type III effector, particularly an E3 ubiquitin ligase (NEL) effector that can activate NOD1 to enhance IL-8 secretion, thus modulating innate immunity.
  • NTL E3 ubiquitin ligase
  • Suitable exemplary primers for generating an sspH2 deletion are provided in Table 7 and correspond to denoted primers including SDP224 through SDP235 and SEQ ID NOs: 178-180, SEQ ID NO: 11, SEQ ID NO:12 and SEQ ID NOs: 181-187, respectively as indicated.
  • a genomic nucleic acid sequence for sspH2 is provided below (SEQ ID NO: 188) >sspH2 AroA, is part of the shikimate pathway, which directly connects glycolysis to the synthesis of aromatic amino acids.
  • SDP484 (5’ TCTAGAAGAAGCTTGGGATCG 3’) (SEQ ID NO: 189) SDP485 (5’ AGCTCTCCCGGGAATTCATG 3’) (SEQ ID NO: 190)
  • SDP486 (5’ CATGAATTCCCGGGAGAGCTGCAGGACTGACGCTGGTTATC 3’) (SEQ ID NO: 191)
  • SDP487 (5’ AACAGAAGACGAAACTCAACTCTCAAAAAACAGAAATAAAAAC 3’)
  • SDP488 (5’ GTTGAGTTTCGTCTTCTGTTGCGCCAGTC 3’) (SEQ ID NO: 193) SDP489 (5’ GATCCCAAGCTTCTTCTAGAATGGCCCAGTTCATGGCC 3’) (SEQ ID NO: 194)
  • SDP486 and SDP487 are used to amplify upstream region of aroA
  • SDP488 and SDP489 are used to amplify the downstream region of aroA
  • SEQ ID NO: 170 represents UP_aroA
  • SEQ ID NO: 171 represents Down_aroA
  • SEQ ID NO: 172 represents pRE112_ ⁇ aroA.
  • AsD ⁇ -aspartate-semialdehyde dehydrogenase
  • L-ASA L-aspartate-semialdehyde
  • Amplified fragments are then joined using overlap extension PCR. Resulting DNA fragment was digested with SacI and KpnI. Digested fragment was ligated with SacI/KpnI digested pRE112 plasmid. Resulting plasmid was introduced into MeganVac1 cells using electroporation. Positive clones are then streaked on tryptone east extract-agar plate supplemented with 15% sucrose and are grown overnight at 30C. Colonies from sucrose plate are then verified for asd deletion by streaking on plate with and without Diaminopimelic acid.
  • OD values and calculated growth rates are calculated from the OD values. The results are depicted in FIGURE 4. OD values and calculated growth rates in doubling time deletion mutants of each of phoPQ, ompR, ssrAB, SPI2, sifA, sseJ, sopF, SP113, sitABCD, feoABC, and mgtRBC and also the MeganVac strain are provided and compared. Mutants with deletion in the genes for phoPQ and ompR display marginally slower growth kinetics in media. Doubling time is reduced in each of phoPQ, ompR, and sopF compared to the MeganVac strain, with slight reduction for ssrAB and feoABC as well versus the MeganVac strain.
  • gentamicin protection assay is performed. Each mutant is grown in LB broth and used to infect 10 5 HD11 macrophage cells at an MOI of 10. After a 30-minute incubation, excess bacteria are killed by adding growth media containing 100 ⁇ g/ml gentamicin. Only bacteria that have not infected the macrophage cells are susceptible to gentamicin as this drug does not cross the plasma membrane. After a two-hour incubation, the macrophage cells are lysed using 0.1% Triton X-100.
  • Diluted lysate is then plated on BHI agar plates and incubated overnight at 37 °C.
  • the number of each mutant bacteria that survive inside macrophage cells is determined by calculating the colony forming unit (CFU) of each mutant and comparing it to that of the parental MEGANVAC strain. The experiment is repeated three times to confirm the results as presented in FIGURE 5.
  • the sitABCD and feoABC deletion mutants displayed the lowest survivability in the macrophage cells.
  • EXAMPLE 4 DELIVERY OF SAM ENCODED GFP USING MUTANTS
  • Mutant Salmonella bacteria deleted for various survival factors as described in Example 3 are used to deliver self-amplifying mRNA encoding GFP as the forign or heterologous oritein or gene of interest.
  • the A69 plasmid encoding GFP (see Example 2) is transformed inside each mutant.
  • Chicken macrophage-like cell line HD11 is seeded in a 24-well plate at a density of 10 5 cells per well.
  • phorbol ester phorbol myristate acetate (PMA) is added at a final concentration of 100 ng/ml.
  • Nanobodies are small, low molecular weight, single-domain, heavy-chain only antibody found in camelids. Owing to its smaller size, genes of these proteins are easy to clone inside a plasmid. Therefore, by using molecular cloning techniques, nanobodies against various antigens can be presented in the systemic circulation.
  • Clostridium perfringens bacteria are associated with severe and significant disease in animals. C. perfringens is associated with hemorrhagic and necrotic enteritis in poultry cattle, sheep, goats and swine.
  • the necrotic enteritis B-like toxin (NetB) is cytotoxic for avian cells and associated with avian necrotic enteritis.
  • a VHH antibody directed against the netb antigen of Clostridium perfringens has been utilized to demonstrate nanobody and single chain, single domain antibodies using the delivery platform.
  • Nucleic acid encoding VHH (SEQ ID NO: 169) was incorporated in the SAM vector.
  • VHH expression was demonstrated utilizing each of MeganVac bacteria, phoPQ mutant bacteria and sifA mutant bacteria ( Figures 7-9).
  • GFP-tagged VHH was also incorporated in the SAM vector and GFP-VHH expression in cells was evaluated by fluorescence microscopy ( Figure 10). Successful antibody expression was demonstrated using the delivery platform.
  • RNA interference [000261]
  • the live intracellular delivery platform can not only introduce foreign genes into host cells but it can also be used to alter host gene expression.
  • One target may be porcine CD163 which functions as the receptor for the Porcine Reproductive and Respiratory Syndrome (PRRS) virus on porcine macrophages.
  • PRRS virus is also known to infect the Marc-145 monkey cell line through CD163 as the receptor.
  • siRNA short interfering RNA
  • RNA interference is a biological mechanism in which small RNA molecules bind to specific mRNA molecules and inhibit translation.
  • shRNA and siRNA against Porcine Reproductive and Respiratory Syndrome (PRRS) viral surface proteins docking CD163/SRCR5 host receptor are designed and cloned into the SAM vector. Resulting plasmids are transformed into Salmonella Typhimurium.
  • PRRS Porcine Reproductive and Respiratory Syndrome
  • Resulting plasmids are transformed into Salmonella Typhimurium.
  • western blot and qPCR is performed from cell extracts of Salmonella infected cell line HEK293T.
  • CD163 levels are evaluated by RT- PCR. Cells are transfected with shRNA and siRNA encoding plasmids.
  • siRNA sequences are provided as follows: siRNA1 (SEQ ID NO:135) [000267] Delivering Antigens as a Therapeutic Vaccine [000268] Delivery of antigens in a vaccine strategy is an important and viable application of the platform provided herein.
  • the delivery platform is utilized to deliver antigens which can serve as immunogenic polypeptides to stimulate an immune reaction and promote immunity, such as immunity against infection by an infectious agent in an animal.
  • Avian coccidosis is a common poultry disease caused by Eimeria.
  • Eimeria is a genus of parasites that includes various species capable of causing the disease coccidiosis in animals such as cattle, poultry, dogs (especially puppies), cats (especially kittens), and smaller ruminants including sheep and goats. Species of this genus infect a wide variety of hosts.
  • the most prevalent species of Eimeria that cause coccidiosis in cattle are E. bovis, E. zuernii, and E. auburnensis.
  • Eimeria infections are particularly damaging to the poultry industry and costs the United States more than $1.5 billion in annual loses.
  • the most economically important species among poultry are E. tenella, E. acervulina, and E. maxima.
  • Salmonella Typhimurium is modified to deliver cross-protective antigens covering Eimeria tenella, E. maxima and E. acervulina as a part of the SAM payload.
  • Eimeria antigens including Eimeria tenella elongation factor -1 ⁇ ; EtAMA1; Eimeria tenella 5401; Eimeria acervulina lactate dehydrogenase antigen gene; Eimeria maxima surface antigen gene; Glyceraldehyde 3-phosphate Dehydrogenase (GAPDH); Eimeria common antigen 14-3-3 are cloned in the self-amplifying nucleic acid vector as the applicable gene of interest. Expression of GAPDH, AMA1 and 14-3-3 Eimeria protein antigens in the delivery platform system using mutant S. typhimurium is depicted in FIGURE 12. [000271] Cocci antigen sequences:
  • Streptococcus suis bacteria is a bacteria that infects Streptococcus suis infects swine (pigs) worldwide and may be zoonotically transmitted to humans with a mortality rate of up to 20%.
  • PlySs2 is a S.suis phage lytic enzyme capable of killing Streptococcus bacteria including S. suis in vitro and in vivo in animal models (Gilmer DB et al (2013) Antimicrob Agents Chemother 57(6):2743-2750; Gilmer DB et al (2017) PLoS ONE 12(1):e0169180).
  • PlySs2 has both broad and high killing activity against multiple serotypes and strains of S. suis, making it a possible tool in the control and prevention of S. suis infections in pigs and humans.
  • Salmonella typhimurium bacteria particularly attenuated bacteria or survival factor mutant bacteria described herein, are modified to deliver Streptococcus suis PlySs2 lytic enzyme as a part of SAM payload assembly.
  • Nucleic acid encoding PlySs2 sequence (SEQ ID NO: 158) is cloned in a SAM payload vector as described herein as the gene of interest. This approach utilizes the Salmonella bacteria to provide systemic bio-pharmaceutical delivery.
  • Bioactive PlySs2 is confirmed utilizing bacterial cell killing assays, including as previously described (Gilmer DB et al (2013) Antimicrob Agents Chemother 57(6):2743-2750).
  • EXAMPLE 6 PLASMID STABILIZATION [000275] To stably maintain a plasmid with self-amplifying mRNA (such as A69) inside Salmonella bacteria, an auxotropic S. typhimurium strain is generated, such that the mutant is unable to synthesize a particular organic compound required for its growth, thus requiring supplemented medium for growth and survival.
  • the asd gene encoding aspartate ⁇ -semialdehyde dehydrogenase, is removed from the chromosome using the same two-step recombination approach as used to construct the survival factor deletion mutants to generate a ⁇ asd strain.
  • a ⁇ asd strain requires an external supply of diaminopimelic acid (DAP) for survival.
  • DAP diaminopimelic acid
  • the same asd gene along with its native promoter is then cloned into the self-amplifying mRNA vector or plasmid (such as that of A69) and is introduced into Salmonella mutants. Once inside the bacteria and DAP is removed from the growth media, the plasmid is indispensable for survival of the bacteria.
  • Biocontainment is an important criterion for any live intracellular delivery platform to ensure any recombinant bacteria shed from the target animal will not survive in the environment.
  • the thyA gene encoding thymidylate synthase is removed from a Salmonella strain. This gene is necessary for DNA synthesis, especially when the bacteria is an environment lacking nucleotides such as outside a host body. Therefore, deletion of thyA in a Salmonella strain results in the death of the mutant bacteria when outside a host organism. Deletion of thyA is accomplished using methods in accordance with those described above in Example 3 for deletion of Salmonella survival factor genes.
  • thyA sequence encodes S. typhimurium protein NP_461918.1 (SEQ ID NO: 166): [000283] Enhanced Delivery via Delayed Lysis [000284] To enhance delivery of the self-amplifying mRNA vector, bacteria need to be killed once inside the host phagocyte. Lysins have been shown to effectively kill bacteria, including as described above. To enhance this, the bacteriophage PhiX174 lysis gene E is cloned into the bacterial chromosome. The promoter of the mgtC gene, which expresses at a very high level once inside the macrophage, directs expression of this gene.
  • the bacteriophage PhiX174 gene E sequence is as follows (SEQ ID NO: 167): [000286]
  • the mgtC gene promoter sequence is as follows (SEQ ID NO: 168): [000287] REFERENCES 1. Kong, W., Clark-Curtiss, J., and Curtiss, R.3rd.2013. Utilizing Salmonella for antigen delivery: the aims and benefits of bacterial delivered vaccination. Expert Reviews in Vaccines. 12(4]:345-7. 2. Kong, W., Brovold, M., Koeneman, B. A., Clark-Curtiss, J., and Curtiss, R. 3rd.2012.
  • RNA Vaccines Give Equivalent Protection against Influenza to mRNA Vaccines but at Much Lower Doses.
  • Salmonella maintains the integrity of its intracellular vacuole through the action of SifA. EMBO Journal 19[13]:3235-3249.
  • Salmonella enterica is one of the most important foodborne pathogens that infects a variety of animals and birds, causing the most common foodbome illnesses in the United States and worldwide, with nearly one-third of the cases attributed to contaminated eggs and poultry.
  • S. Typhi and S. Paratyphi A causes the typhoid fever.
  • S. Typhimurium causes gastroenteritis, leading to vomiting, diarrhea, fever, and abdominal cramps.
  • Vaccination has proven to be an effective strategy to reduce Salmonella load in poultry.
  • Attenuated Salmonella has been used as a vaccine in poultry providing protection against Salmonellosis.
  • attenuated strains of Salmonella can be used as an oral delivery vector for biotherapeutics in animals and potentially in humans.
  • the Salmonella Typhimurium ⁇ crp- ⁇ cya (MeganVacl) strain is the most commonly used vaccine in the United States; however further understanding of the mechanisms of virulence attenuation and host response to this vaccine strain would be relevant.
  • Salmonella According to the Center for Disease Control and Prevention, every year in the United States alone, Salmonella causes serious infections in about 1.35 million people, among them 26,500 people need hospitalization, and 420 people die (National Center for Emerging and Zoonotic Infectious Diseases (NCEZID), 2020; WHO, 2020).
  • NCEZID National Center for Emerging and Zoonotic Infectious Diseases
  • the Salmonella genus is comprised of 2 species, Salmonella enterica and Salmonella bongori, and includes more than 2600 known serotypes (Lin-Hui Su 2007, Michael P. Ryan 2017).
  • S. Typhimurium and S. Enteriridis are the predominant ones that infect humans and result in gastroenteritis with diarrhea, fever and abdominal cramps as the primary symptoms.
  • Salmonella Although uncommon, invasive infections with life-threatening complications with bacteremia and extra-intestinal manifestations can occur in risk populations such as infants, young children, the elderly and immunocompromised patients (Gordon 2008).
  • Salmonellosis Several serotypes have been associated with Salmonellosis, with S. Enteritidis as the most frequent one followed by S. Typhimurium (Su and Chui, 2007; Ryan et al., 2017).
  • Salmonella has a broad host range and colonizes the gastrointestinal tract of a variety of domestic and wild animals, including poultry, without any detectable symptoms (Barrow et al., 2012). Humans usually contract Salmonella by consuming contaminated food of animal origin, mainly through contaminated poultry meat, eggs, and milk.
  • Salmonella As an intercellular pathogen with a broad host range, Salmonella has evolved to survive in very harsh conditions. After ingestion, Salmonella first encounters the highly acidic gastric juice inside the stomach.
  • Salmonella upregulates several amino acid decarboxylase systems (de Jonge , Park 1996, Kieboom 2006, Morita 2006, A ⁇ lvarez-Ordo ⁇ n ⁇ ez 2010) and induces synthesis of acid shock proteins including RpoS and PhoPQ (Audia 2001, Tu 2006). Changes in the composition of cell membrane fatty acid and the resulting changes in membrane fluidity also play a vital role in the survival of Salmonella in low pH (A ⁇ lvarez-Ordo ⁇ n ⁇ ez 2008, Alonso-Hernando 2010). Furthermore, exposure to low pH increases the virulence of Salmonella (Gahan 1999).
  • Salmonella Once Salmonella reaches the gut, it encounters bile which possesses strong antimicrobial properties (Begley 2005, Merritt 2009). Salmonella is inherently resistant to bile due to the upregulation of genes encoding two-component signal transduction systems, efflux pumps, and various transcriptional regulators (Begley 2005). Salmonella also upregulates several genes to survive in high salt and low oxygen environments of the intestine (Frymier 1997, Wei 1999, Sevc ⁇ ⁇ k 2001, Balaji 2005, Su 2009). In the intestine, Salmonella invades intestinal epithelial cells and dendritic cells and induces its own uptake by antigen sampling M cells; of these, uptake via M cells is the preferred route for Salmonella.
  • Salmonella uses its long polar fimbriae to attach to M cells (B. D. Jones 1994, Bäumler A.J.1996) and induces membrane ruffles, which engulf the bacteria, resulting in endocytosis (Jepson M.A.1998).
  • Salmonella has the ability to invade intestinal epithelial cells using both the trigger mechanism (Type III secretion system-dependent) and zipper mechanism (Type III secretion system-independent) (McGhie et ak, 2009, Rosselin 2011, Moest and Meresse 2013). Salmonella can also modify the intestinal epithelial cells into M cells, thus promoting its own uptake (Amin Tahoun 2012).
  • the M cells are located on top of the Payer’s patches, and actively transport bacteria to underlying macrophages.
  • Salmonella Once Salmonella enters macrophages, it resides inside a modified endosome known as Salmonella Containing Vacuole (SCV). Inside this SCV, Salmonella alters the expression of a plethora of genes to adapt itself to survive and replicate (Alpuche-Aranda CM 1994, Meresse S 2001, Srikumar S 2015). The low magnesium and low iron concentration inside SCV activate the PhoP/Q system.
  • SCV Salmonella Containing Vacuole
  • This two-component system then upregulates the expression of mgtRBC and feoABC operons, which leads to increased uptake of Mg 2+ and Fe 2+ (Groisman 2001, Eunna Choi 2009).
  • the deficiency of manganese inside the SCV also induces the expression of the ABC type transporter sitABCD (David G. Kehres 2002, Ikeda JS 2005).
  • the sensor kinase, EnvZ senses the acidic environment of SCV and engages OmpR to upregulate the expression of various pH regulatory genes (Chakraborty S 2015).
  • OmpR also upregulates the expression of the two-component system SsrAB (Lee AK 2000).
  • SsrAB acts as a master regulator and upregulates the expression of genes in Salmonella Pathogenicity Island 2 (SPI2) (Worley MJ 2000, Walthers D 2007).
  • SPI2 encodes for the Type III secretion system that delivers at least 28 effector molecules responsible for SCV maturation and maintenance inside macrophages (Figueira R 2012, Fàbrega A 2013).
  • effectors several of them are required for SCV maturation and maintenance (eg. SIfA, sopF, SseJ), bacterial growth and replication (eg. SteA), host-cell modification (eg. SpvB, SteC), and inhibiting host immune response (eg.
  • Poultry products are one of the major sources of Salmonella outbreaks in western countries (WHO 2002, Ray LC 2021). Consumption of undercooked eggs, egg products, and meat are one of the main contributing factors in sporadic Salmonella infections (Hayes, Nylen et al.1999, Kimura, Reddy et al.2004).
  • Antibiotics have been used successfully to eliminate Salmonella from the food chain, but the emergence of antibiotic-resistant Salmonella has become a major concern in public food safety (Felicita Medalla 2021). As a viable alternative, vaccination is likely to take a focal position in fighting against Salmonella.
  • Several strategies have been developed to generate attenuated Salmonella that induces a strong immune response when administered orally (Erova, Kirtley et al.2016). Attenuated Salmonella have also been used to orally deliver heterologous antigens leading to both mucosal and systemic immune response (Lin, Van et al.2015, Yurina 2018). However, knowledge about the host cell response against the invasion of these attenuated mutants is largely unknown.
  • the S. Typhimurium crp-cya mutant (MeganVacl; also referred to here as ⁇ crp-cya) is the first and the most commonly used live attenuated vaccine in the United States (Curtiss R, 3rd, Hassan JO. Vet Immunol Immunopathol. l 996;54(1-4):365-72; Curtiss, R., 3rd and S. M. Kelly (1987). Infection and immunity 55(12): 3035-3043; Hassan JO, Curtiss R, 3rd. Res Microbiol.1990;141(7-8):839-50; Hassan JO, Curtiss R, 3rd.
  • HD11 cell line and culture conditions [000301] HD11 cells were procured from US Department of Agriculture (USDA).
  • USDA US Department of Agriculture
  • HD11 is a chicken macrophage-like cell line that was derived from chicken hematopoietic cells after in vitro transformation with the avian myelocytomatosis type MC29 virus (Beug, von Kirchbach et al.1979).
  • HD11 cells were maintained in Iscove Modified Dulbecco Media (IMDM) medium (Gibco) supplemented with 10% fetal bovine serum (FBS) at 39°C inside a humidified 5% CO 2 incubator.
  • IMDM Iscove Modified Dulbecco Media
  • FBS fetal bovine serum
  • This strain carries a cya-crp deletion that makes it avirulent but invasive (Curtiss and Kelly 1987).
  • the ⁇ cya-crp mutant (MeganVac1) was constructed from the pathogenic S. Typhimurium strain UK-1 (Luo et al, 2011). This strain (UK-1) was purchased from ATCC (ATCC 68169) and used as a control. To construct additional Salmonella deletion mutants, altered in other Salmonella genes, the ⁇ -red recombinase- mediated recombination was used (Figure 11) (Datsenko and Wanner 2000).
  • the whole cassette was then amplified using PCR and electroporated into the ⁇ cya-crp mutant cells harboring the ⁇ -red recombinase encoding plasmid, pSIJ8 (Jensen et al, 2015).
  • the expression of ⁇ -red recombinase was induced by adding 50 mM of arabinose.
  • ⁇ cya-crp mutant cells were maintained at 30°C. After two hours of incubation at 30°C, the transformants were plated on a BHI-agar plate supplemented with 50 ⁇ g/ml chloramphenicol. Successful transformants were verified using PCR and Sanger sequencing.
  • the pSIJ8 plasmid was then cured by sub-culturing the mutants at 42°C overnight for five passages. Depletion of the plasmid was verified by carbenicillin sensitivity test (pSIJ8 has a functional bla gene) followed by lack of PCR amplification of the plasmid backbone.
  • carbenicillin sensitivity test pSIJ8 has a functional bla gene
  • lack of PCR amplification of the plasmid backbone [000306]
  • Growth kinetics of Salmonella mutants [000307] Salmonella mutants were grown overnight in BHI medium supplemented with 0.1% glucose at 37°C while shaking.
  • PCN Phosphate Carbon Nitrogen
  • the PCN media contained 80 mM morpholineethanesulfonic acid (MES), pH 4.8, 4 mM Tricine, 100 ⁇ M FeCl 3 , 376 ⁇ M K 2 SO 4 , 50 mM NaCl, 0.4 mM K 2 HPO 4 /KH 2 PO 4 , pH 4.8, 0.4% glucose, 15 mM NH4Cl, 1 mM MgSO4 and 10 ⁇ M CaCl2.
  • MES morpholineethanesulfonic acid
  • pH 4.8 Tricine
  • a cocktail of micronutrients was also added to the media.
  • the composition of micronutrients was 10 nM Na2MoO4.2H2O, 10 nM NaSeO3, 4 nM H3BO3, 300 nM CoCl2.6H2O, 100 nM CuSO4.5H2O, 800 nM MnCl2 and 1nM ZnSO4.. Final pH was adjusted to 5.8.
  • the growth of all the Salmonella mutants was monitored using SpectraMax®i3x multi-plate reader as described above.
  • Carbon source utilization assays [000309] The carbon source utilization of the Salmonella mutants was investigated using PM1 and PM2A MicroPlateTM (Biolog Inc.). The plates were set up according to the manufacturer’s protocol.
  • the bacterial cells were grown overnight on BHI-agar plates. Single colonies were then streaked on BHI-agar plates covering the whole surface. The culture was then scraped with a sterile cotton swab and resuspended in 5 ml of inoculation fluid (IF0) until turbidity (T) of 42% is reached. Three ml of 42% T cell suspension was then mixed with 15 ml IF0+dye. One hundred microliters of cell suspension was then added into each well of the PM1 and PM2A plates. Plates were then incubated at 37C overnight. Cell growth was analyzed by visual inspection of color change.
  • IF0 inoculation fluid
  • Invasion and intracellular survival assay [000311] Chicken macrophage-like cell line HD11 was maintained in IMDM (Gibco) medium supplemented with 10% FBS (Gibco). The day before infection, approximately 1 x 10 5 cells were seeded in each well of a 24 well plate and incubated at 39°C under 5% CO2 atmosphere. HD11 cells were activated by phorbol 12-myristate 13-acetate (PMA) at a concentration of 100 ng/mL (Wisner, Potter et al.2011). Overnight grown Salmonella mutants were sub-cultured and grown until they reached an OD 600 0f 1.0.
  • PMA phorbol 12-myristate 13-acetate
  • Bacterial cells were then washed twice in DPBS and resuspended in IMDM medium supplemented with 10% heat-inactivated FBS.
  • HD11 cells were also washed twice with DPBS and the bacteria were added to each well at a multiplicity of infection (MOI) of 10.
  • MOI multiplicity of infection
  • the plates were centrifuged at 600Xg for 5 minutes. The plates were then incubated at 39 ⁇ C for 30 minutes. After infection, the extracellular bacteria were removed by washing twice with DPBS and replacing the medium with fresh complete IMDM medium supplemented with 100 ⁇ g/mL gentamicin.
  • PBMCs Chicken peripheral blood mononuclear cells
  • PBMCs Chicken peripheral blood mononuclear cells
  • Tubes were then centrifuged at 400 X g for 30 minutes at room temperature with the lowest acceleration and no braking. After centrifugation, the top plasma layer was removed and the interface was collected in a fresh tube. The collected lymphocytes were then mixed gently with 5 volumes of DPBS and centrifuged at 250 X g for 10 minutes at room temperature. The supernatant was discarded, and the pellet was washed twice with DPBS at room temperature.
  • the pellet was then finally resuspended in RPMI 1640 Medium, supplemented with GlutaMAXTM (ThermoFisher Sc.), 10% fetal bovine serum (FBS) (Gibco), Anti- anti (ThermoFisher Sc.) and 50 ⁇ g/ml chicken granulocyte-macrophage colony stimulating factor (GMCSF) (Abcam).
  • GlutaMAXTM ThermoFisher Sc.
  • FBS fetal bovine serum
  • Anti- anti ThermoFisher Sc.
  • GMCSF chicken granulocyte-macrophage colony stimulating factor
  • Bacterial cells were then washed twice in DPBS and resuspended in 1 ml RPMI 1640 medium with 10% heat-inactivated FBS.
  • the primary macrophages were infected with the Salmonella mutant strains with an MOI of 10 for 30 minutes. After 30 minutes, extracellular bacterial were killed by adding 100 ⁇ g/ml gentamicin for an hour, and the medium was then replaced with fresh RPMI-1640 medium supplemented with 30 ⁇ g/ml gentamicin and incubated for another hour.
  • Cells were then washed twice with DPBS and lysed by using the RLT buffer (Qiagen).
  • RNA samples with RNA integrity number (RIN) > 8.0, A260/A280 > 1.9 and A260/A230 > 2 were selected for mRNA library preparation and Next Generation Sequencing. Briefl, the total RNA was incubated with mRNA capture beads to remove contaminating ribosomal RNA from the sample using the Kapa Stranded mRNA-Seq kit (Kapa Biosystems) following manufacturer’s instructions. The resulting poly(A)-captured mRNA was then fragmented.
  • First-strand cDNA synthesis was performed using reverse transcriptase and random primers in the presence of Actinomycin D, followed by second-strand cDNA synthesis with DNA polymerase I and RNase H. Double-stranded cDNA was end-repaired and A-tailed for subsequent adaptor ligation. Indexed adaptors were ligated to the A-tailed cDNA. Enrichment by PCR was performed to generate the final cDNA sequencing library. Libraries were sequenced as paired-end 150 base reads on an Illumina NextSeq500 following the manufacturer's protocols. [000317] Reads were aligned, quantified and analyzed using CLC Genomics Workbench (Qiagen).
  • the ⁇ crp-cya mutant is one of the most commonly used and successful live attenuated vaccines used in the United States to reduce Salmonella load in poultry. Salmonella has evolved several strategies to successfully evade host defense mechanisms within macrophages. Previous studies have identified several key Salmonella factors required for survival within macrophages. To better understand the infection phenotypes and early response of macrophages to the ⁇ crp-cya mutant and its derivatives lacking key factors required for intra-macrophage survival, we generated S.
  • Salmonella deletion mutants were created by ⁇ -red recombinase-mediated recombination.
  • the MeganVac1 cells were first electroporated with the ⁇ -red recombinase expression plasmid pSIJ8 (Jensen, Lennen et al.2015) and transformants were selected based on carbenicillin sensitivity of the transformed cells. Salmonella carrying the plasmid was maintained at 30 ⁇ C throughout the experiment.
  • PCR amplification was performed to verify the deletions.
  • phoPQ ompR-envZ, ssrAB, SP113, sitABC, feoABC, mgtRBC and sspH2, primers were designed flanking the GOIs.
  • SPI2 sifA, sopF and sseJ, one primer was inside the GOI and the other outside of it.
  • the pSIJ8 plasmid was cured by incubating the bacteria at 42 ⁇ C for five passages.
  • in-vitro characterization of Salmonella mutants [000324] We first tested the fitness of these mutants by growing them in a defined media (M9MM + glucose).
  • ⁇ crp-cya mutant and its derivatives were unable to metabolize sugars or substrates that are used in the TCA cycle (succinic acid, malic acid, ⁇ - ketoglutaric acid, citric acid, fumaric acid), in the pentose phosphate pathway (D-glucuronic Acid, D-Xylose, D-Ribose, Lactulose, Sucrose, ⁇ -Hydroxy Glutaric Acid- ⁇ Lactone) and glyoxylate pathway (glyoxylic acid).
  • the ⁇ crp-cya mutant and its derivatives were also unable to metabolize amino acids that convert into TCA cycle intermediates (L-Aspartic Acid, L-Proline, L-Glutamic Acid, L-Asparagine, L-Glutamine, L- Histidine and L- Alanine) and carbon sources that lead to TCA cycle intermediates (acetic acid, propionic acid, Glycolic Acid, Glyoxylic Acid and Glycyl-L Glutamic Acid).
  • ⁇ crp-cya mutant compared to ⁇ crp-cya mutant, ⁇ crp-cya- ⁇ ssrAB, ⁇ crp-cya- ⁇ SPI2, ⁇ crp-cya- ⁇ SPI13, ⁇ crp-cya- ⁇ mgtRBC, ⁇ crp-cya- ⁇ sopF, ⁇ crp-cya- ⁇ sitABCD, ⁇ crp-cya- ⁇ sseJ, and ⁇ crp-cya- ⁇ sspH2 displayed increased ability to invade macrophages.
  • the three mutants lacking three master regulators, MeganVac1 (cya, crp), phoPQ and ompR-envZ showed a significant reduction in CFU count (3.2, 6.2, and 16-fold respectively).
  • the fourth master regulator ssrAB displayed increased CFU compared to the MeganVac1 strain.
  • ssrAB directly regulates the expression of the genes of SPI2.
  • SPI2 deletion mutants showed a similar CFU to that of ssrAB deletion strain. Deletion of SPI 13 also resulted in greater invasion by Salmonella.
  • deletion of feoABC resulted in a significant reduction in the invasion of Salmonella.
  • ⁇ crp-cya mutant compared to ⁇ crp-cya mutant, ⁇ crp-cya- ⁇ ssrAB, ⁇ crp-cya- ⁇ SPI2, ⁇ crp-cya- ⁇ SPI13, ⁇ crp-cya- ⁇ mgtRBC, ⁇ crp-cya- ⁇ sopF, ⁇ crp-cya- ⁇ sitABCD, ⁇ crp-cya- ⁇ sseJ, and ⁇ crp-cya- ⁇ sspH2 displayed increased ability to survive inside macrophages.
  • PBMCs were isolated from healthy chicken and cultured in the presence of chicken GMCSF to differentiate into macrophages.
  • the macrophages were monitored for their differentiation using an inverted microscope ( Figure 15A).
  • PBMCs were attached to the well surface and the floaters were removed from the culture and a fresh medium containing GMCSF was added. After three days, the adherent cells became flat and demonstrated classical mammalian M1 macrophage-like morphology (Peng, van den Biggelaar et al.2020). After five days the majority of cells were differentiated into macrophages.
  • NetworkAnalysit program was used to perform pathway enrichment using the over-representation analysis option. It was also used to visualize the network of differentially expressed genes (Xia, Benner et al.2014). Approximately, 80–86% of the reads mapped to the reference genome. Of these reads, 69.8–74.2% of the reads mapped to exonic regions, 14.2–18.8% mapped to intronic regions and 11.5–12.6% mapped to intergenic regions. The coefficients of determination (R2) between replicates and samples ranged from 0.98 to 0.99.
  • transcripts whose expression was significantly altered in Salmonella infected cells (fold change of ⁇ 2 or ⁇ -2, with an adjusted p-value of 0.1).
  • a total of 127 transcripts were differentially expressed in UK1 infected macrophages. Among them 49 transcripts were more than 2-fold over-expressed and 75 transcripts were down-regulated more than 2-fold.
  • the significantly upregulated genes belong to NOD-like receptor signaling pathway, Toll-like receptor signaling pathway, Cytosolic DNA-sensing pathway, RIG-I-like receptor signaling pathway, Cytokine-cytokine receptor interaction, Apoptosis, and Ubiquitin mediated proteolysis (P value ⁇ 0.05).
  • a total of 84, 217, 118 and 52 transcripts were differentially expressed in MeganVac1, phoPQ, sifA, and sopF respectively.
  • DEGs between UK1 and MeganVac1 only four genes (CCL5, REL, TRAF3, and KRT23) were upregulated and twelve genes (ANKRD9, TTYH2, DIPK2A, LFNG, GPR34, NEK2, EPB41, LY75, TNFAIP8, MARCO, ARAP3, and ENSGALG00000029035) were down-regulated in both UK1 and MeganVac1 infected macrophages.
  • the down- regulated genes belong to the mTOR signaling pathway, cell cycle progression, autophagy, lysosome biogenesis, lipid metabolism, p53 signaling, ABC transporter-mediated ion transport, cellular senescence, and RAS signaling.
  • mTOR signaling pathway cell cycle progression
  • autophagy autophagy
  • lysosome biogenesis lipid metabolism
  • p53 signaling ABC transporter-mediated ion transport
  • cellular senescence and RAS signaling.
  • RAS signaling a similar set of pathways were significantly upregulated in phoPQ, sifA, and sopF infected macrophages.
  • These genes belong to Toll-like receptor signaling, cytokine signaling, NOD-like receptor signaling, Cytosolic DNA sensing, MAPK signaling, and apoptosis.
  • Gene ontology (GO) over-representation analysis using Innate DB suggested significant upregulation of biological processes such as chromosome condensation, metal ion transport, superoxide metabolic process, Golgi vesicle transport, intracellular signal transduction, regulation of gene expression, and carbohydrate biosynthesis (Table 5).
  • a similar pattern was observed for downregulated pathways, which consist of p53 signaling, amino sugar, and nucleotide sugar metabolism, ABC transporter-mediated ion transport, biotin metabolism, Galactose metabolism, and cellular senescence.
  • GO over-representation analysis also revealed significant down-regulation of Glycosphingolipid metabolic process, carbohydrate metabolism, antigen processing and presentation, inflammatory response, organ morphogenesis (Table 6).
  • cya-crp, phoPQ, ssrAB and ompR-EnvZ are global transcriptional regulators that are activated soon after the internalization of S. Typhimurium by macrophages. These regulators then activate a plethora of genes downstream. Activation of ssrAB leads to the activation of Salmonella pathogenicity island (SPI) 2 and 13 which in turn activates the expression of effectors such as sifA, sopF and sspH2.
  • SPI Salmonella pathogenicity island
  • Ion transporters such as mgtRBC, feoABC and sitABCD help to maintain homeostasis inside the SCV.
  • the mutant strains exhibited significant changes in their phenotypic characters such as their growth kinetics, doubling time, and caron utilization. In rich media, the majority of the mutants showed significant increases in doubling times compared to the UK1 strain. Interestingly, the doubling time of phoPQ decreased significantly compared to both the MeganVac1 and the UK1 strain. However, significant reduction in doubling time was observed for all the mutants in PCN media. The lag phase was also delayed in all the mutants. The PCN media mimics the SCV condition.
  • the mutant strains exhibited similar growth characteristic to that of MeganVac1, suggesting no significant growth defects were manifested upon further deletion of these genes.
  • the UK1 strain demonstrated viability in the presence of a wide range of carbon sources. Whereas the MeganVac1 and its derivative mutants could not metabolize a majority of the sugars utilized in TCA, making these mutants efficient auxotrophs.
  • all the mutants, but not the UK1 strains exhibited significant growth defects in BHI broth without the added glucose (data not shown). This auxotrophic property of these mutants makes them suitable candidates for vaccines or as a delivery vector.
  • Salmonella has a robust intracellular lifestyle, where they invade macrophages and reside inside a specialized compartment known as SCV. This allows Salmonella to survive and replicate while it remains protected from the macrophage attack.
  • SCV a specialized compartment
  • Salmonella enterica subspecies enterica particularly S. Enteritidis and S. Typhimurium, are one of the most common causes of foodbome illnesses worldwide, with majority of the illnesses attributed to consumprion of contaminated poultry meat and eggs. Vaccination has been proven to be an effective strategy to reduce Salmonella burden in poultry. The S.
  • Typhimurium crp-cya mutant is one of the widely used vaccines in the United States; however, the infection phenotypes and the early macrophage response to this vaccine strain are relatively poorly understood.
  • ⁇ crp-cya and its derivatives ⁇ crp-cya- ⁇ phoPQ, ⁇ crp-cya- ⁇ ompR-envZ, ⁇ crp-cya- ⁇ feoABC and ⁇ crp- cya- ⁇ SifA had greater doubling times in SCV-simulating media and were highly attenuated for invasion and intracellular survival within macrophages; the derivatives were more attenuated than ⁇ crp-cya mutant.
  • ⁇ crp-cya derivatives lacking ssrAB, SPIJ 3, SPI2, mgtRBC, sitABCD, sopF, sseJ and sspH2 had greater doubling times but surprisingly showed increased invasion and intracellular survival compared to ⁇ crp-cya mutant.
  • Transcriptome analyses of macrophages infected with parent strain, ⁇ crp- cya mutant and its derivatives lackingphaPQ, sifA and sopF demonstrated that similar changes in gene expression were observed in macrophages infected with these strains.
  • the differentially upregulated genes primarily belonged to innate immunity, immunoregulation, cellular homeostasis and response to pathogens.
  • Cyclic AMP receptor protein (CRP) and adenylate cyclase (Cya) are two global regulators required for bacterial response to carbon starvation and these genes have been deleted in the crp-cya mutant.
  • CRP Cyclic AMP receptor protein
  • Cya adenylate cyclase
  • the crp-cya mutant and majority of its derivatives exhibited increased doubling times.
  • the vaccine strain derivatives ⁇ crp-cya- ⁇ ompR-envZ, ⁇ crp-cya- ⁇ SopF and ⁇ crp-cya- ⁇ SspH2 showed decreased doubling times than the ⁇ crp-cya strain but similar doubling times as the parent strain.
  • ⁇ crp-cya- ⁇ phoPQ strain had decreased doubling time than both the crp-cya mutant and its parent strain. The reasons for these unusual phenotypes are unknown. [000351] Consistent with the fact that the carbon starvation response genes crp and cya are deleted in the vaccine strain, the crp-cya mutant and its derivatives exhibited significant defects in carbon source utilization. As expected, the parent strain demonstrated the ability to utilize a wide range of carbon sources. However, the crp-cya mutant and its derivative were unable to metabolize the majority of carbon sources related to TCA cycle. All the mutants strains also exhibited significant growth defects in BHI broth without the added glucose (data not shown).
  • the ⁇ crp-cya mutant and its derivatives lacking phoPQ, ompR-envZ, sifA and feoABC exhibited severe growth defects and increased doubling times in an SCV-simulating media and were highly attenuated for invasion and intracellular survival within macrophages. All the four ⁇ crp-cya mutant derivatives were more attenuated than the mutant strain itself; of all the strains tested in this study, the derivatives lacking ompR-envZ and feoABC showed the highest attenuation.
  • the cya-crp system represses the expression of SPIl, which is required for invasion (69).
  • PhoPQ is a global regulator and regulates the expression of a number of downstream genes upon sensing the acidic environment of the SCV (70, 71). PhoPQ activates grhDJ, which plays an important role in S. Typhimurium invasion (72). PhoPQ protects S. Typhimurium against reactive nitrogen species by regulating intracellular Mg2+ concentration (73). PhoPQ-activated genes also protect S. Typhimurium from antimicrobial peptides produced by macrophages (74-76) and the acidic environment of the SCV (77). The importance of OmpR-EnvZ in S. Typhimurium invasion and survival has previously been demonstrated in an S. Typhi infection model (78).
  • OmpR-EnvZ activates the ssrAB two-component system which in turn induces the expression of SPI2 genes (79-82).
  • SifA is an effector of T3SS-2 and plays a key role in maintaining the integrity of SCV and formation of Salmonella-induced tubules and is required for Salmonella virulence (83, 84).
  • FeoABC is required for uptake of ferrous iron and help Salmonella maintain iron homeostasis inside SCV (85, 86).
  • ssrAB leads to activation of Salmonella pathogenicity island (SPI) 2 and 13, which in turn activate expression of effectors such as SopF, SspH2 and SseJ (80, 87).
  • Ion transporters such as MgtRBC and SitABCD help Salmonella maintain ion homeostasis inside SCV (88-90).
  • Salmonella infection is characterized by a marked global rearrangement of the macrophage transcriptome (91, 92).
  • Transcriptome profiling of macrophages infected with UK.I, ⁇ crp-cya and its derivatives lacking phoPQ, sifA and sopF demonstrated that, compared to unifected macrophages, 138, 148, 153, 155 and 142 genes were differentially expressed in these strains, respectively.
  • the macrophage response to the ⁇ crp-cya mutant was similar to that of its parent strain UK.I.
  • TLRs are upregulated in response to bacterial lipopolysaccharides (93, 94). Activation of TLR signaling pathway cascades into the activation of NFK ⁇ pathway. Activated NFKB in turn induces the transcription of several genes that are involved in differentiation, inflammation, and cell survival (95-97).
  • the NOD-like receptors (NLRs) also play an important role in pathogen recognition and signal induction of downstream genes.
  • NLRs are intracellular sensors that mainly recognize intracellular bacteria (98). NLRs including both Nodl and Nod2 recognize bacterial peptidoglycans and cascade the signal to induce the activation of NFKB and MAPK pathway (99-101). Signaling thorough NLRs results in a strong inflammatory response via secretion of proinflammatory cytokines (102-104); consistent with this, several genes encoding chemokines and cytokines were also upregulated in response to Salmonella infection.
  • RIG-I is another cytosolic pathogen sensing system that senses short dsRNA and sRNA; infection with UK.I, the crp-cya mutant its derivatives induced upregulation of genes in the RIG-I-like signalling patway.
  • FIGURE 18 A schematic diagram showing the molecular mechanisms of macrophage response to Salmonella infection is shown in FIGURE 18.
  • Modifications in membrane fatty acid composition of Salmonella typhimurium in response to growth conditions and their effect on heat resistance 123, 212–219.
  • Hassan JO, Curtiss R, 3rd Effect of vaccination of hens with an avirulent strain of Salmonella typhimurium on immunity of progeny challenged with wild-Type Salmonella strains. Infect Immun.1996;64(3):938-44. Hayes, S., G. Nylen, R. Smith, R. L. Salmon and S. R. Palmer (1999). "Undercooked hens eggs remain a risk factor for sporadic Salmonella enteritidis infection.” Commun Dis Public Health 2(1): 66-67. Huang, K., A. Herrero-Fresno, I. Th ⁇ fner, S. Skov and J. E.
  • Salmonella T3SSs successful mission of the secret(ion) agents. 16: 38–44. Saliba, A.-E., L. Li, A. J. Westermann, S. Appenzeller, D. A. C. Stapels, L. N. Schulte, S. Helaine and J. Vogel (2016). "Single-cell RNA-seq ties macrophage polarization to growth rate of intracellular Salmonella.” Nature Microbiology 2(2): 16206. Seemann, T. (2014). "Prokka: rapid prokaryotic genome annotation.” Bioinformatics 30(14): 2068-2069. Sevc ⁇ ⁇ k, M., Sebkova ⁇ , A., Volf, J.
  • Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction. J Biol Chem.1999;274(16):10689-92. 94. Yang RB, Mark MR, Gray A, Huang A, Xie MH, Zhang M, et al. Toll-like receptor-2 mediates lipopolysaccharide-induced cellular signalling. Nature.1998;395(6699):284-8. 95. Baeuerle PA, Henkel T. Function and activation ofNF-kappa Bin the immune system. Annual review of immunology.1994;12:141-79. 96. Barkett M, Gilmore TD. Control of apoptosis by Rel/NF-KB transcription factors. Oncogene.
  • Girardin SE Boneca IG, Carneiro LA, Antignac A, Jehanno M, Viala J, et al. Nodl detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science (New York, NY).2003;300(5625):1584- 7. 101. Hayden MS, Ghosh S. Signaling to NF-kappaB. Genes & development.2004;18(18):2195-224. 102. Travassos LH, Carneiro LA, Girardin SE, Boneca IG, Lemos R, Bozza MT, et al. Nodl participates in the innate immune response to Pseudomonas aeruginosa.

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Abstract

La présente invention concerne une plateforme d'administration intracellulaire comprenant une bactérie atténuée portant un acide nucléique auto-amplifiant codant pour une biomolécule thérapeutique. La biomolécule thérapeutique a une activité ou un effet souhaité tel que le traitement ou la prévention d'une infection, d'une maladie ou d'un trouble ou l'induction d'une immunité protectrice contre un organisme pathogène ou parasitaire. L'invention concerne des bactéries génétiquement modifiées et atténuées, en particulier des bactéries Salmonella. L'invention concerne des procédés d'administration intracellulaire et systémique d'une ou de plusieurs biomolécules thérapeutiques. L'invention concerne des acides nucléiques auto-amplificateurs.
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