US20080124355A1 - Live bacterial vaccines for viral infection prophylaxis or treatment - Google Patents

Live bacterial vaccines for viral infection prophylaxis or treatment Download PDF

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US20080124355A1
US20080124355A1 US11/859,569 US85956907A US2008124355A1 US 20080124355 A1 US20080124355 A1 US 20080124355A1 US 85956907 A US85956907 A US 85956907A US 2008124355 A1 US2008124355 A1 US 2008124355A1
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salmonella
antigen
enterica serovar
bacterial
salmonella enterica
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David Gordon Bermudes
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AVIEX TECHNOLOGIES LLC
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Priority to US11/859,569 priority Critical patent/US20080124355A1/en
Priority to AU2007300519A priority patent/AU2007300519A1/en
Priority to CN200780043473A priority patent/CN101720228A/zh
Priority to EP07838725A priority patent/EP2081593A4/fr
Priority to RU2009115177/15A priority patent/RU2009115177A/ru
Priority to PCT/US2007/020578 priority patent/WO2008039408A2/fr
Priority to CA2700218A priority patent/CA2700218A1/fr
Publication of US20080124355A1 publication Critical patent/US20080124355A1/en
Assigned to AVIEX TECHNOLOGIES, LLC reassignment AVIEX TECHNOLOGIES, LLC ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BERMUDES, DAVID GORDON
Assigned to AVIEX TECHNOLOGIES LLC reassignment AVIEX TECHNOLOGIES LLC ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BERMUDES, DAVID G., PH.D
Priority to US13/369,333 priority patent/US8440207B2/en
Priority to US13/892,380 priority patent/US9315817B2/en
Priority to US15/131,083 priority patent/US10087451B2/en
Priority to US16/148,055 priority patent/US10626403B2/en
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    • 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
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • 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
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
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    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01018—Exo-alpha-sialidase (3.2.1.18), i.e. trans-sialidase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • 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
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • A—HUMAN NECESSITIES
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    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • C—CHEMISTRY; METALLURGY
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    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • This invention is generally in the field of live bacterial vaccines for viral infection prophylaxis or treatment.
  • Influenza viruses There are three types of influenza viruses Influenza A, B, and C.
  • Influenza types A or B viruses cause epidemics of disease almost every winter. In the United States, these winter influenza epidemics can cause illness in 10% to 20% of people and are associated with an average of 36,000 deaths and 114,000 hospitalizations per year.
  • Influenza type C infections cause a mild respiratory illness and are not thought to cause epidemics.
  • Influenza type A viruses are divided into subtypes based on two proteins on the surface of the virus. These proteins are termed hemagglutinin (H) and neuraminidase (N). Influenza A viruses are divided into subtypes based on these two proteins.
  • H3N2 viruses have been found in the population. Because H1N1 viruses returned in 1977, two influenza A viruses are presently cocirculating (Palese and Garcia-Sarstre J Clin Invest, July 2002, Volume 110, Number 1, 9-13). The pathogenicity of the initial 1918 H1N1 has not been equaled by any of the latter H1N1, H2N2 or H3N2 subtypes, although infection from some subtypes can be severe and result in death. By molecular reconstruction, the genome of the 1918 flu including the amino acid sequences of the H1 and N1 antigens is now known (Kaiser, Science 310: 28-29, 2005; Tumpey et al., Science 310: 77-81, 2005).
  • the optimum way of dealing with a human pandemic virus would be to provide a clinically approved well-matched vaccine (i.e., containing the hemagglutinin and/or neuraminidase antigens of the emerging human pandemic strain), but this cannot easily be achieved on an adequate timescale because of the time consuming method of conventional influenza vaccine production in chicken eggs.
  • a clinically approved well-matched vaccine i.e., containing the hemagglutinin and/or neuraminidase antigens of the emerging human pandemic strain
  • Live attenuated bacterial vaccine vectors offer an important alternative to conventional chicken egg based vaccines. Growth on embryonated hen eggs, followed by purification of viruses from allantoic fluid, is the method by which influenza virus has traditionally been grown for vaccine production. More recently, viruses have been grown on cultured cell lines, which avoids the need to prepare virus strains that are adapted to growth on eggs and avoids contamination of the final vaccine with egg proteins. However, because some of the vaccine virus may be produced in canine tumor cells (e.g., MDCK), there is concern for contamination of the vaccine by cancer causing elements. Moreover, both must undergo a labor intensive and technically challenging purification process, with a total production time of 3 to 6 months.
  • canine tumor cells e.g., MDCK
  • Antibody responses are typically measured by enzyme linked immunosorbent assay (ELISA), immunoblotting, hemagglutination inhibition, by microneutralisation, by single radial immunodiffusion (SRID), and/or by single radial hemolysis (SRH). These assay techniques are well known in the art.
  • Salmonella bacteria have been recognized as being particularly useful as live “host” vectors for orally administered vaccines because these bacteria are enteric organisms that, when ingested, can infect and persist in the gut (especially the intestines) of humans and animals.
  • Salmonella bacteria As a variety of Salmonella bacteria are known to be highly virulent to most hosts, e.g., causing typhoid fever or severe diarrhea in humans and other mammals, the virulence of Salmonella bacterial strains toward an individual that is targeted to receive a vaccine composition must be attenuated. Attenuation of virulence of a bacterium is not restricted to the elimination or inhibition of any particular mechanism and may be obtained by mutation of one or more genes in the Salmonella genome (which may include chromosomal and non-chromosomal genetic material).
  • an “attenuating mutation” may comprise a single site mutation or multiple mutations that may together provide a phenotype of attenuated virulence toward a particular host individual who is to receive a live vaccine composition for avian influenza.
  • a variety of bacteria and, particularly, serovars of Salmonella enterica have been developed that are attenuated for pathogenic virulence in an individual (e.g., humans or other mammals), and thus proposed as useful for developing various live bacterial vaccines (see, e.g., U.S. Pat. Nos.
  • Bacterial flagella are known to be antigenic and subject to antigenic or phase variation which is believed to help a small portion of the bacteria in escaping the host immune response.
  • the bacterial flagellar antigens are referred to as the H1 and H2 antigens.
  • the bacterial flagellar H antigen will be referred to as fH henceforth. Because the Salmonella -based vaccination of a heterologous antigen is dependent upon the bacteria's ability to colonize the gut, which may be reduced do to the initial immune response, the vaccination ability of the second immunization may be diminished due to an immune response to the vector.
  • Hin invertase belongs to the recombinase family, which includes Gin invertase from phage Mu, Cin invertase from phage P1, and resolvases from Tn3 and the transposon (Glasgow et al. 1989, p. 637-659. In D. E. Berg, and M. M. Howe (ed.), Mobile DNA. American Society for Microbiology, Washington, D.C.). Hin promotes the inversion of a chromosomal DNA segment of 996 bp that is flanked by the 26-bp DNA sequences of hixL and hixR (Johnson and Simon. 1985. Cell 41:781-791). Hin-mediated DNA inversion in S.
  • Hin 21 kDa exists in solution as a homodimer and binds to hix sites as a dimer (Glasgow et al. 1989. J. Biol. Chem. 264:10072-10082).
  • a cis-acting DNA sequence recombinational enhancer
  • F binding protein
  • Live Salmonella vaccines have not had deletions of the hin gene nor defined all or fH2 antigens, nor have they been constructed such that they lack fH antigens altogether. Accordingly, live Salmonella vaccines have not been constructed to maximize a prime-boost strategy which alternates or eliminates the fH antigen whereby the immune response of the fH antigen of the first immunization (prime) is not specific for the anigen of the second immunization (boost). Therefore, the boost immunization is not diminished by a rapid elimination by the immune system, and is therefore able to persist longer and more effectively present the immunizing antigen.
  • prime-boost strategy which alternates or eliminates the fH antigen whereby the immune response of the fH antigen of the first immunization (prime) is not specific for the anigen of the second immunization (boost). Therefore, the boost immunization is not diminished by a rapid elimination by the immune system, and is therefore able to persist longer and more effectively present
  • GEMs genetically engineered microorganisms
  • Such genes could in theory provide virulence factors to non-pathogenic or less pathogenic viral strains if allowed to recombine under circumstances were the bacterial vaccine could co-occur at the same time in the same individual as a wild type viral infection.
  • methods that reduce bacterial recombination and increase bacterial genetic isolation are desirable.
  • IS200 elements are genetic elements that can insert copies of themselves into different sites in a genome. These elements can also mediate various chromosomal rearrangements, including inversions, deletions and fusion of circular DNA segments and alter the expression of adjacent genes. IS200 elements are found in most Salmonella species. S. typhimurium strain LT2 has six IS200s. Salmonella typhimurium strain 14028 has been described to possess an additional IS200 element at centisome 17.7 which is absent in other commonly studied Salmonella strains LT2 and SL1344 (Murray et al., 2004 Journal of Bacteriology, 186: 8516-8523). These authors describe a spontaneous hot spot (high frequency) deletion of the Cs 17.7 to Cs 19.9 region. Live Salmonella vaccines have not had deletions of IS200 elements which would limit such recombination events.
  • Salmonella strains are known to possess phage and prophage elements. Such phage are often capable of excision and infection of other susceptible strains and are further capable of transferring genes from one strain by a process known as transduction. Live Salmonella vaccines have not had deletions in phage elements such as phage recombinases which exist in Salmonella , such that the phage are no longer capable of excision and reinfection of other susceptible strains.
  • Salmonella strains are known to be capable of being infected by bacteria phage. Such phage have the potential to carry genetic elements from one Salmonella strain to another. Live Salmonella vaccines have not comprised mechanisms to limit phage infection such as the implantation and constitutive expression of the P22 phage repressor C2.
  • Veiga et al. 2003 Journal of Bacterilogy 185: 5585-5590
  • hybrid proteins containing the b-autotransporter domain of the immunogloulin A (IgA) protease of Nisseria gonorrhoea demonstrated hybrid proteins containing the b-autotransporter domain of the immunogloulin A (IgA) protease of Nisseria gonorrhoea.
  • IgA immunogloulin A
  • Kahn et al. (EP No. 0863211) have suggested use of a live bacterial vaccine with in vivo induction using the E. coli nitrite reductase promoter nirB. These authors further suggest that the antigenic determinant may be an antigenic sequence derived from a virus, including influenza virus.
  • Khan et al. did not describe a vaccine for avian influenza virus. They did not describe the appropriate antigens for an avian influenza virus, the hemagluttinin and neuraminidase, and did not describe how to genetically match an emerging avian influenza virus.
  • live avian influenza vaccines would not be genetically isolated or have improved genetic stability in order to provide a live vaccine for avian influenza that would be acceptable for use in humans.
  • Khan et al. state that any of a variety of known strains of bacteria that have an attenuated virulence may be genetically engineered and employed as live bacterial carriers (bacterial vectors) that express antigen polypeptides to elicit an immune response including attenuated strains of S. typhimurium and, for use in humans, attenuated strains of S. typhi (i.e., S. enterica serovar Typhi).
  • non-reverting mutations especially deletion mutations which provide the attenuation.
  • non-reversion only refers to the particular gene mutated, and not to the genome per se with its variety of IS200, phage and prophage elements capable of a variety of genetic recombinations and/or even transductions to other bacterial strains.
  • Khan et al. did not describe a bacterial strain with improved genetic stability, nor methods to reduce genetic recombination, such as deletion of the IS200 elements.
  • Khan et al. did not describe a bacterial strain with improved genetic stability by deletion of the bacteria phage and prophage elements nor limiting their transducing capacity. Neither did Khan et al. describe methods to minimize bacterial genetic exchange, such as constitutive expression of the P22 C2 phage repressor.
  • the present invention provides live attenuated bacterial strains that express one or more immunogenic polypeptide antigens of a virus, preferably an avian influenza virus, that is effective in raising an immune response in mammals.
  • a virus preferably an avian influenza virus
  • one aspect of the invention relates to live attenuated bacterial strains which may include Salmonella vectoring avian influenza antigens that can be administered orally to an individual to elicit an immune response to protect the individual from avian influenza.
  • the preferred bacteria are serovars of Salmonella .
  • the bacteria are genetically isolated from infecting bacteria phage and have improved genetic stability by virtue of deletion of IS200 and phage elements.
  • the preferred Salmonella strains of the invention are attenuated by mutations at genetic loci which, alone or in combination, provides sufficient attenuation, and defined flagellar antigens for improved an improved prime/boost strategy.
  • the attenuating mutations may be those of strains known to exhibit a degree of safety in humans including but not limited to Ty21a, CMV906, CMV908, CMV906-htr, CMV908-htr, Ty800, holavax, M01ZH09 or VNP20009 or may be novel combinations of mutations.
  • the current medical practice uses derivatives of pathogenic avian strains in chicken eggs to provide vaccines that generate an immune response including antibodies in humans or other mammals against known pathogenic avian strains
  • the invention provides methods and compositions comprising genetically isolated bacterial vectors with enhanced genetic stability vectoring avian influenza virus antigens to protect against emerging pathogenic human strains.
  • the invention targets viruses for vaccine strains based on their emerging pathogenicity, and produces an effective vaccine more closely matched to the antigen profile of the emerging pathogen.
  • the invention requires detailed knowledge of the antigenic profile of an emerging strain, such a vaccine can be produced at the time of need in order to reduce the risk of an unmatched vaccine and potential effects of partial protection in a human pandemic outbreak.
  • the invention provides vaccines for protecting a human patient against infection by an emerging avian influenza virus strain.
  • the vaccines according to the present invention comprise genetically stable bacterial vectors carrying one or more antigen from an avian influenza virus strain that can cause highly pathogenic avian influenza.
  • the invention further preferably provides for vaccines against oseltamivir resistant strains.
  • a live Salmonella bacterial vaccine in accordance with the present invention, that is genetically engineered to express one or more avian influenza antigens as described herein have the inherent ability to establish a population (infection) in the gut and, if properly modified they could provide a desirable source of immunogenic avian influenza antigen polypeptide(s) to elicit an immune response in the mucosal tissue of the individual.
  • the antigen(s) can invoke an antibody response in the patient that is capable of neutralizing the emerging avian influenza vaccine strains with high efficiency, as well as emerging heterologous avian influenza vaccine strains, with moderate efficiency.
  • the emerging avian influenza vaccine will be within the same hemagglutinin and or neuraminidase type (i.e., H1, H5, H5 (H274Y), H7 or H9 and/or N1, N2 or N7) as are the current pathogenic avian influenza strains.
  • the live vaccine compositions are suitable for oral administration to an individual to provide protection from avian influenza.
  • a vaccine composition comprises a suspension of a live bacterial strain described herein in a physiologically accepted buffer or saline solution that can be swallowed from the mouth of an individual.
  • oral administration of a vaccine composition to an individual may also include, without limitation, administering a suspension of a bacterial vaccine strain described herein through a nasojejunal or gastrostomy tube and administration of a suppository that releases a live bacterial vaccine strain to the lower intestinal tract of an individual.
  • Vaccines of the invention may be formulated for delivery by other various routes e.g. by intramuscular injection, subcutaneous delivery, by intranasal delivery (e.g.
  • intradermal delivery e.g. WO02/074336, WO02/067983, WO02/087494, WO02/0832149 WO04/016281
  • Injection may involve a needle (including a microneedle), or may be needle-free.
  • Annual human influenza vaccines typically include more than one influenza strain, with trivalent vaccines being normal (e.g. two influenza A virus antigens, and one influenza B virus antigen). In pandemic years, however, a single monovalent strain may be used.
  • the pathogenic avian antigen(s) described above may be the sole influenza antigen(s) in a vaccine of the invention, or the vaccine may additionally comprise antigen(s) from one or more (e.g. 1, 2, 3, 4 or more) annual influenza virus strains.
  • Specific vaccines of the invention thus include: (i) a vaccine comprising the pathogenic avian antigen(s) as the sole influenza antigen(s); (ii) a vaccine comprising the pathogenic avian antigen(s) plus antigen(s) from another pathogenic avian influenza strain (e.g., H1N1, H5N1, H7N7, H2N9, H9N2).
  • a vaccine comprising the pathogenic avian antigen(s) plus antigen(s) from another pathogenic avian influenza strain (e.g., H1N1, H5N1, H7N7, H2N9, H9N2).
  • Vaccines of the invention use one or more avian antigens to protect patients against infection by an influenza virus strain that is capable of human-to-human transmission i.e. a strain that will spread geometrically or exponentially within a given human population without necessarily requiring physical contact.
  • the patient may also be protected against strains that infect and cause disease in humans, but that are caught from birds rather than from other humans (i.e., bird to human transmission).
  • the invention is particularly useful for protecting against infection by pandemic, emerging pandemic and future pandering human strains e.g. for protecting against H5 and N1 influenza subtypes.
  • the invention may protect against any hemagglutinin subtypes, including H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16 or various neuraminidase subtypes, including N1, N2, N3, N4, N5, N6, N7, N8 or N9.
  • the characteristics of an influenza strain that give it the potential to cause a pandemic outbreak may include: (a) it contains a new or antigenically altered hemagglutinin compared to the hemagglutinins in currently-circulating human strains i.e., one that has not been evident in the human population for over a decade (e.g. H2), or has not previously been seen at all in the human population (e.g.
  • H5, H6 or H9 that have generally been found only in bird populations), such that the human population will be immunologically naive to the strain's hemagglutinin or that is a subtype which is antigenically altered by changes in amino acid sequence or glycosylation; (b) it is capable of being transmitted horizontally in the human population; (c) is capable of being transmitted from animals (including birds, dogs, pigs) to humans; and/or (d) it is pathogenic to humans.
  • one embodiment of the invention in accordance with that aspect will generally include at least one gene that originated in a mammalian (e.g. in a human) influenza virus and one gene which originated in a bird or non-human vertibrate.
  • Vaccines in accordance with various aspects of the invention may therefore include an antigen from an avian influenza virus strain. This strain is typically one that is capable of causing highly pathogenic avian influenza (HPAI).
  • HPAI is a well-defined condition (Alexander Avian Dis (2003) 47(3 Suppl):976-81) that is characterized by sudden onset, severe illness and rapid death of affected birds/flocks, with a mortality rate that can approach 100%.
  • Low pathogenicity (LPAI) and high pathogenicity strains are easily distinguished e.g. van der Goot et al. (Epidemiol Infect (2003) 131(2):1003-13) presented a comparative study of the transmission characteristics of low and high pathogenicity H5N2 avian strains.
  • examples of HPAI strains are H 5 N1 influenza A viruses e.g.
  • the avian influenza strain may be of any suitable hemagglutinin subtype, including H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16.
  • the avian influenza strain may further be of any suitable neuraminidase subtype N1, N2, N3, N4, N5, N6, N7, N8, or N9.
  • the vaccines of the invention may comprise two or more (i.e., two, three, four, or five) avian influenza hemagglutinin and neuraminidase antigens.
  • avian influenza strains may comprise the same or different hemagglutinin subtypes and the same or different neuraminidase subtypes.
  • a preferred vaccine composition will contain a sufficient amount of live bacteria expressing the antigen(s) to produce an immunological response in the patient. Accordingly, the attenuated Salmonella strains described herein are both safe and useful as live bacterial vaccines that can be orally administered to an individual to provide immunity to avian influenza and, thereby, protection from avian influenza.
  • an effective mucosal immune response to avian influenza antigen(s) in humans by oral administration of genetically engineered, attenuated strains of Salmonella strains as described herein may be due to the ability of such mutant strains to persist in the intestinal tract.
  • Each bacterial strain useful in the invention carries an antigen-expressing plasmid or chromosomally integrated cassette that encodes and directs expression of one or more avian influenza antigens of avian influenza virus when resident in an attenuated Salmonella strain described hererin.
  • avian influenza antigens that are particularly useful in the invention include an H1, H5, H5 (H274Y), H7 or H9 antigen polypeptide (or immunogenic portion thereof), a N1, N2 or N7 antigen polypeptide (or immunogenic portion thereof), and a fusion polypeptide comprising a heterologous secretion peptide linked in-frame to the antigenic peptide.
  • the serovars of S. enterica that may be used as the attenuated bacterium of the live vaccine compositions described herein include, without limitation, Salmonella enterica serovar Typhimurium (“S. typhimurium”), Salmonella montevideo, Salmonella enterica serovar Typhi (“S. typhi”), Salmonella enterica serovar Paratyphi B (“S. paratyphi 13”), Salmonella enterica serovar Paratyphi C (“S. paratyphi C”), Salmonella enterica serovar Hadar (“S. hadar”), Salmonella enterica serovar Enteriditis (“S. enteriditis”), Salmonella enterica serovar Kentucky (“S.
  • Salmonella enterica serovar Infantis (“S. infantis”), Salmonella enterica serovar Pullorurn (“S. pullorum”), Salmonella enterica serovar Gallinarum (“S. gallinarum”), Salmonella enterica serovar Muenchen (“S. muenchen”), Salmonella enterica serovar Anatum (“S. anatum”), Salmonella enterica serovar Dublin (“S. dublin”), Salmonella enterica serovar Derby (“S. derby”), Salmonella enterica serovar Choleraesuis var. kunzendorf (“S. cholerae kunzendorf’), and Salmonella enterica serovar minnesota (S. minnesota).
  • live avian influenza vaccines in accordance with aspects of the invention include known strains of S. enterica serovar Typhimurium (S. typhimurium) and S. enterica serovar Typhi (S. typhi) which are further modified as provided by the invention to form suitable vaccines for the prevention and treatment of avian influenza.
  • Such Strains include Ty21a, CMV906, CMV908, CMV906-htr, CMV908-htr, Ty800, aroA-/serC-, holavax, M01ZH09, VNP20009.
  • Novel strains are also encompassed that are attenuated in virulence by mutations in a variety of metabolic and structural genes.
  • the invention therefore may provide a live vaccine composition for protecting against avian influenza comprising a live attenuated bacterium that is a serovar of Salmonella enterica comprising, an attenuating mutation in a genetic locus of the chromosome of said bacterium that attenuates virulence of said bacterium and wherein said attenuating mutation is the Suwwan deletion (Murray et al., 2004) or combinations with other known attenuating mutations.
  • Attenuating mutation useful in the Salmonella bacterial strains described herein may be in a genetic locus selected from the group consisting of phoP, phoQ, edt, cya, crp, poxA, rpoS, htrA, nuoG, pmi, pabA, pts, damA, purA, purB, purl, zwf, purF, aroA, aroB, aroC, aroD, serC, gua, cadA, rfc, rjb, rfa, ompR, msbB and combinations thereof.
  • the invention may also provide a process for preparing genetically stable bacterial vaccines for protecting a human patient against infection by a avian influenza virus strain, comprising genetically engineering the avian antigen from an avian influenza virus strain that can cause highly pathogenic avian influenza to comprise a bacterially codon optimized expression sequence within a bacterial plasmid expression vector or chromosomal localization expression vector and further containing engineered restriction endonuclease sites such that the bacterially codon optimized expression gene contains subcomponents which are easily and rapidly exchangeable in order to facilitate rapid exchange of the genetic subcomponents to achieve a well matched antigen to the emerging avian influenza pathogen.
  • the plasmid and/or chromosomal expression constructs may be further modified to result in the secretion of the viral antigens.
  • Administration of the vaccine to the patient invokes an antibody response that is capable of neutralizing said avian influenza virus strain.
  • the invention may also provide methods and compositions for producing a bacterial vector expressing one or more avian influenza antigens where said bacterial vector has one or more deletions in IS200 elements which results in enhance genetic stability.
  • the composition and methods comprise a bacterial strain with a deletion in the IS200 elements, such that the bacteria are no longer capable of genetic rearrangement using IS200 elements. Such a deletion is generated in any one or more IS200 element, which is then confirmed using standard genetic techniques.
  • the invention may also provide methods and compositions for producing a genetically stabilized bacterial vector expressing one or more avian influenza antigens where said bacterial vector has one or more deletions in bacteria phage or prophage elements which enhanced genetic stability and prevent phage excision.
  • the composition and methods comprise a bacterial strain with one or more deletions in bacteria phage or prophage elements, such that the bacteria are no longer capable of genetic rearrangement using bacteria phage or prophage elements. Such a deletion is generated in any bacteria phage or prophage elements, which is then confirmed using standard genetic techniques. Such strains have phage with reduced capacity for transduction of genes to other strains.
  • the invention may also provide methods and compositions for producing a bacterial vector expressing one or more avian influenza antigens where said bacterial vector constitutively expresses the P22 phage C2 repressor, thereby preventing new infections by bacteria phage and further preventing subsequent phage transductions by these phage.
  • Live Salmonella vaccines have not had deletions of the hin gene nor defined fH1 or fH2 antigens, nor have they been constructed such that they lack fH antigens altogether.
  • prior live Salmonella vaccines have not been constructed to maximize a prime-boost strategy which alternates or eliminates the fH antigen whereby the immune response of the fH antigen of the first immunization (prime) is not specific for the anigen of the second immunization (boost). Therefore, the boost immunization is not diminished by a rapid elimination by the immune system, and is therefore able to persist longer and more effectively present the immunizing heterologous avian influenza antigen.
  • An embodiment of the present invention therefore may also provide methods and compositions for producing a bacterial vector expressing one or more avian influenza antigens where said bacterial vector has a defined flagellar H antigen (fH).
  • the composition and methods comprise a bacterial strain with a deletion in the Hin recombinase gene, such that the bacteria are no longer capable of alternating between fH1 and fH2 antigens. Such a deletion is generated in either an fH1 or fH2 serologically defined strain, which is then reconfirmed following deletion or disruption of the hin recombinase gene.
  • the invention further provides methods and compositions for producing a bacterial vector which lacks flagellar antigens generated by deletion of the fliBC genes (i.e., fH0). Therefore, an improved composition for a prime/boost strategy is provided where the second vaccination comprises administration of a vaccine where the fH antigen composition is different from the first vaccination.
  • the invention may also provide a method for protecting a human patient against infection by an avian influenza virus strain with an improved prime/boost strategy, comprising the step of administering to the patient a vaccine that comprises an antigen from an avian influenza virus strain that can cause highly pathogenic avian influenza or 1918 influenza within a bacterial vector expressing one or more avian influenza antigens where said bacterial vector has a defined fH antigen or no fH antigen (i.e., fH1, fH2, or fH0).
  • the invention may further provide a method of administering a second bacterial vector expressing one or more avian influenza antigens comprising a second step where the second administration where said bacterial vector has a defined fH antigen which is different fH antigen composition than the fH antigen of the first administration or no fH antigen.
  • the second administration includes a bacterial vaccine where the first vaccine administration is a bacterial vaccine of the present invention or is another vaccine not encompassed by the present application, e.g., another bacterial vaccine or an egg-based vaccine.
  • the invention may also provide a kit comprising (a) a first container comprising a bacterial expression codon optimized antigen from a pathogenic avian influenza virus strain containing unique genetically engineered restriction sites contained within either a bacterial protein expression plasmid or a bacterial chromosomal protein expression vector and (b) a second container comprising bacterial vector(s) with one or more (e.g., fH1, fH2 or fH0) flagellar antigen(s).
  • Component (a) will be modifiable to genetically match an emerging avian influenza virus using standard in vitro molecular techniques and can be combined with component (b) to generate one or more bacterial strains with defined flagellar antigens which constitute a live vaccine.
  • the variation(s) in flagellar antigens provided by the kit provide for more than one live vaccine strain in which a first immunization (prime) using one strain may be followed at an appropriate time such as 2 to 4 weeks by a second immunization (boost) using a second strain with a different fH antigen or no fH antigen.
  • the live vaccine compositions are suitable for oral administration to an individual to provide protection from avian influenza.
  • a vaccine composition comprises a suspension of a live bacterial strain described herein in a physiologically accepted buffer or saline solution that can be swallowed from the mouth of an individual.
  • oral administration of a vaccine composition to an individual may also include, without limitation, administering a suspension of a bacterial vaccine strain described herein through a nasojejunal or gastrostomy tube and administration of a suppository that releases a live bacterial vaccine strain to the lower intestinal tract of an individual.
  • Attenuated refers to elimination or reduction of the natural virulence of a bacterium in a particular host organism, such as a mammal. “Virulence” is the degree or ability of a pathogenic microorganism to produce disease in a host organism. A bacterium may be virulent for one species of host organism (e.g., a mouse) and not virulent for another species of host organism (e.g., a human). Hence, broadly, an “attenuated” bacterium or strain of bacteria is attenuated in virulence toward at least one species of host organism that is susceptible to infection and disease by a virulent form of the bacterium or strain of the bacterium.
  • the term “genetic locus” is a broad term and comprises any designated site in the genome (the total genetic content of an organism) or in a particular nucleotide sequence of a chromosome or replicating nucleic acid molecule (e.g., a plasmid), including but not limited to a gene, nucleotide coding sequence (for a protein or RNA), operon, regulon, promoter, regulatory site (including transcriptional terminator sites, ribosome binding sites, transcriptional inhibitor binding sites, transcriptional activator binding sites), origin of replication, intercistronic region, and portions therein.
  • a genetic locus may be identified and characterized by any of a variety of in vivo and/or in vitro methods available in the art, including but not limited to, conjugation studies, crossover frequencies, transformation analysis, transfection analysis, restriction enzyme mapping protocols, nucleic acid hybridization analyses, polymerase chain reaction (PCR) protocols, nuclease protection assays, and direct nucleic acid sequence analysis.
  • infection has the meaning generally used and understood by persons skilled in the art and includes the invasion and multiplication of a microorganism in or on a host organism (“host”, “individual”, “patient”) with or without a manifestation of a disease (see, “virulence” above).
  • Infectious microorganisms include pathogenic viruses, such as avian influenza, that can cause serious diseases when infecting an unprotected individual.
  • An infection may occur at one or more sites in or on an individual.
  • An infection may be unintentional (e.g., unintended ingestion, inhalation, contamination of wounds, etc.) or intentional (e.g., administration of a live vaccine strain, experimental challenge with a pathogenic vaccine strain).
  • a site of infection includes, but is not limited to, the respiratory system, the alimentary canal (gut), the circulatory system, the skin, the endocrine system, the neural system, and intercellular spaces.
  • replication of infecting microorganisms comprises, but is not limited to, persistent and continuous multiplication of the microorganisms and transient or temporary maintenance of microorganisms at a specific location.
  • an “infection” of a host individual with a live vaccine comprising genetically altered, attenuated Salmonella bacterial strain as described herein is desirable because of the ability of the bacterial strain to elicit a protective immune response to antigens of avian influenza virus that cause avian influenza in humans and other mammals.
  • disease and “disorder” have the meaning generally known and understood in the art and comprise any abnormal condition in the function or well being of a host individual.
  • a diagnosis of a particular disease or disorder, such as avian influenza, by a healthcare professional may be made by direct examination and/or consideration of results of one or more diagnostic tests.
  • a “live vaccine composition”, “live vaccine”, “live bacterial vaccine”, and similar terms refer to a composition comprising a strain of live Salmonella bacteria that expresses at least one antigen of avian influenza, e.g., the H antigen, the N antigen, or a combination thereof, such that when administered to an individual, the bacteria will elicit an immune response in the individual against the avian influenza antigen(s) expressed in the Salmonella bacteria and, thereby, provide at least partial protective immunity against avian influenza.
  • Such protective immunity may be evidenced by any of a variety of observable or detectable conditions, including but not limited to, diminution of one or more disease symptoms (e.g., respiratory distress, fever, pain, diarrhea, bleeding, inflammation of lymph nodes, weakness, malaise), shorter duration of illness, diminution of tissue damage, regeneration of healthy tissue, clearance of pathogenic microorganisms from the individual, and increased sense of well being by the individual.
  • a disease symptoms e.g., respiratory distress, fever, pain, diarrhea, bleeding, inflammation of lymph nodes, weakness, malaise
  • diminution of tissue damage e.g., regeneration of healthy tissue, clearance of pathogenic microorganisms from the individual, and increased sense of well being by the individual.
  • a live vaccine comprising a bacterium described herein may be, at the discretion of a healthcare professional, administered to an individual who has not presented symptoms of avian influenza but is considered to be at risk of infection or is known to already have been exposed to avian influenza virus, e.g., by proximity or contact with avian influenza patients or virally contaminated air, liquids, or surfaces.
  • oral refers to administration of a compound or composition to an individual by a route or mode along the alimentary canal.
  • oral routes of administration of a vaccine composition include, without limitation, swallowing liquid or solid forms of a vaccine composition from the mouth, administration of a vaccine composition through a nasojejunal or gastrostomy tube, intraduodenal administration of a vaccine composition, and rectal administration, e.g., using suppositories that release a live bacterial vaccine strain described herein to the lower intestinal tract of the alimentary canal.
  • recombinant is used to describe non-naturally altered or manipulated nucleic acids, cells transformed, electroporated, or transfected with exogenous nucleic acids, and polypeptides expressed non-naturally, e.g., through manipulation of isolated nucleic acids and transformation of cells.
  • the term “recombinant” specifically encompasses nucleic acid molecules that have been constructed, at least in part, in vitro using genetic engineering techniques, and use of the term “recombinant” as an adjective to describe a molecule, construct, vector, cell, polypeptide, or polynucleotide specifically excludes naturally existing forms of such molecules, constructs, vectors, cells, polypeptides, or polynucleotides.
  • Cassette, or expression cassette is used to describe a nucleic acid sequence comprising (i) a nucleotide sequence encoding a promoter, (ii) a first unique restriction enzyme cleavage site located 5′ of the nucleotide sequence encoding the promoter, and (iii) a second unique restriction enzyme cleavage site located 3′ of the nucleotide sequence encoding the promoter.
  • the cassette may also contain a multiple cloning site (MCS) and transcriptional terminator within the 5′ and 3′ restriction endonuclease cleavage sites.
  • the cassette may also contain cloned genes of interest.
  • salmonella (plural, “salmonellae”) and “ Salmonella ” refers to a bacterium that is a serovar of Salmonella enterica .
  • Salmonella enterica serovar Typhimurium (“S. typhimurium”) and serovar Typhi (“ S. typhi ”) as described herein.
  • Salmonella enterica serovar Typhimurium (“S. typhimurium”)
  • S. typhi ” serovar Typhi
  • strainstrain and “isolate” are synonymous and refer to a particular isolated bacterium and its genetically identical progeny. Actual examples of particular strains of bacteria developed or isolated by human effort are indicated herein by specific letter and numerical designations (e.g. strains Ty21a, CMV906, CMV908, CMV906-htr, CMV908-htr, Ty800, holavax, M01ZH09, VNP20009).
  • This invention provides live vaccine compositions for protecting against avian influenza comprising live Salmonella enterica serovars that are genetically engineered to express one or more avian influenza antigen polypeptides, such as the H1, H5, H5 (H274Y), H7 or H9 and N1, N2 and N7 antigens of avian influenza virus.
  • live vaccine compositions for protecting against avian influenza comprising live Salmonella enterica serovars that are genetically engineered to express one or more avian influenza antigen polypeptides, such as the H1, H5, H5 (H274Y), H7 or H9 and N1, N2 and N7 antigens of avian influenza virus.
  • FIG. 1 shows a modified ptrc99a plasmid.
  • FIGS. 2A and B show a plasmid vectors capable of expressing the H5 or N1 antigens cytoplasmically.
  • FIG. 3 shows modified ptrc99a plasmid with unique restriction sites engineered into the coding sequence of the N1 gene for rapid exchange of mutations such as the H274Y.
  • FIG. 4A shows a plasmid vectors expressing the H5 or N1 antigens in a secreted form as fusions with the hlyA protein.
  • FIG. 4B shows a plasmid vector expressing HlyB and HlyD genes necessary for secretion of HlyA and HlyA fusion proteins.
  • FIG. 5 shows a plasmid vector for expression of an antigen (e.g., H5 or N1) as a ClyA fusion.
  • an antigen e.g., H5 or N1
  • FIG. 6 shows a plasmid vector for expression of an antigen (e.g., H5 or N1) as a autotransporter fusion.
  • an antigen e.g., H5 or N1
  • FIG. 7 shows a plasmid vector for expression of an antigen (e.g., H5 or N1) as a colicin E3 fusion.
  • an antigen e.g., H5 or N1
  • FIG. 8A shows selection of 5′ and 3′ DNA segments for constructing a pCVD442 chromosomal integration vector.
  • 8B shows the vector for disrupting chromosomal genes and capable of integration of new genes into the chromosome.
  • FIG. 9 shows a cloning sequence, from A) synthetic gene expression vector to B) chromosomal localization vector.
  • FIG. 10 shows a PCR method for determination of IS200 17.7 and 19.9 rearrangement/deletion.
  • FIG. 11 shows a method to achieve the Suwwan deletion in strains lacking the 17.7 Cs IS200.
  • the present invention is based upon a combination of bacterial vector and protein expression technology which results in a unique vaccine which is rapidly constructed in response to emerging avian influenza and their highly pathogenic derivatives.
  • the present invention is directed to the construction bacterially codon optimized avian and human influenza genes and their incorporation into a Salmonella strain for therapeutic use in the prevention of avian influenza and highly pathogenic derivatives.
  • An antigen-expressing plasmid or chromosomal construct in the bacterial strains described herein may also contain one or more transcriptional terminators adjacent to the 3′ end of a particular nucleotide sequence on the plasmid to prevent undesired transcription into another region of the plasmid or chromosome.
  • transcription terminators thus serve to prevent transcription from extending into and potentially interfering with other critical plasmid functions, e.g., replication or gene expression.
  • transcriptional terminators that may be used in the antigen-expressing plasmids described herein include, but are not limited to, the T1 and T2 transcription terminators from 5S ribosomal RNA bacterial genes (see, e.g., FIGS. 1-5 ; Brosius and Holy, Proc. Natl. Acad. Sci. USA, 81: 6929-6933 (1984); Brosius, Gene, 27(2): 161-172 (1984); Orosz et al., Eur. J. Biochem., 20 (3): 653-659 (1991)).
  • the mutations in an attenuated bacterial host strain may be generated by integrating a homologous recombination construct into the chromosome or the endogenous Salmonella virulence plasmid (Donnenberg and Kaper, 1991; Low et al. (Methods in Molecular Medicine, 2003)).
  • a suicide plasmid is selected for integration into the chromosome by a first homologous recombination event, followed by a second homologous recombination event which results in stable integration into the chromosome.
  • the antigen-expressing chromosomal integration constructs described herein comprise one or more nucleotide sequences that encode one or more polypeptides that, in turn, comprise one or more avian influenza antigens, such as the hemagglutinin and neuraminidase polypeptide antigens, or immunogenic portions thereof, from avian influenza virus and highly pathogenic derivatives.
  • avian influenza antigens such as the hemagglutinin and neuraminidase polypeptide antigens, or immunogenic portions thereof, from avian influenza virus and highly pathogenic derivatives.
  • Such coding sequences are operably linked to a promoter of transcription that functions in a Salmonella bacterial strain even when such a bacterial strain is ingested, i.e., when a live vaccine composition described herein is administered orally to an individual.
  • enteric bacteria such as Escherichia coli and serovars of S. enterica (see, e.g., Dunstan et al., Infect. Immun., 67(10): 5133-5141 (1999)).
  • Promoters (P) that are useful in the invention include, but are not limited to, well known and widely used promoters for gene expression such as the naturally occurring Plac of the lac operon and the semi-synthetic Ptrc (see, e.g., Amman et al., Gene, 25 (2-3): 167-178 (1983)) and Ptac (see, e.g., Aniann et al., Gene, 69(2): 301-315 (1988)), as well as PpagC (see, e.g., Hohmann et al., Proc. Natl. Acad. Sci. USA, 92.
  • promoters for gene expression such as the naturally occurring Plac of the lac operon and the semi-synthetic Ptrc (see, e.g., Amman et al., Gene, 25 (2-3): 167-178 (1983)) and Ptac (see, e.g., Aniann et al., Gene, 69
  • PpmrH see, e.g., Gunn et al., Infect. Immun., 68: 6139-6146 (2000)
  • PpmrD see, e.g., Roland et al., J. Bacteriol., 176: 3589-3597 (1994)
  • PompC see, e.g., Bullifent et al., Vacccine, 18: 2668-2676 (2000)
  • PnirB see, e.g., Chatfield et al., Biotech. (NY), 10: 888-892 (1992)
  • PssrA see, e.g., Lee et al., J. Bacteriol. 182.
  • promoters are known to be regulated promoters that require the presence of some kind of activator or inducer molecule in order to transcribe a coding sequence to which they are operably linked. However, some promoters may be regulated or inducible promoters in E.
  • trc trc promoter
  • Ptrc functions as an inducible promoter in Escherichia coli (e.g., using the inducer molecule isopropyl-p-D-1 8 thio-galactopyranoside, “IPTG”), however, in Salmonella bacteria having no Lacd repressor, Ptrc is an efficient constitutive promoter that readily transcribes avian influenza antigen-containing polypeptide coding sequences present on antigen-expressing plasmids described herein. Accordingly, such a constitutive promoter does not depend on the presence of an activator or inducer molecule to express an antigen-containing polypeptide in a strain of Salmonella.
  • the avian influenza antigen-expressing chromosomal integration constructs which integrate into the live vaccine strains also contain an origin of replication (ori) that enables the precursor plasmids to be maintained as multiple copies in certain the bacterial cells which carry the lamda pir element.
  • an origin of replication ori
  • a number of multi-copy plasmids that replicate in Salmonella bacteria are known in the art, as are various origins of replications for maintaining multiple copies of plasmids.
  • Preferred origins of replications for use in the multi-copy antigen-expressing plasmids described herein include the origin of replication from the multi-copy plasmid pBR322 (“pBR ori”; see, e.g., Maniatis et al., In Molecular Cloning: A Laboratoly Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1982), pp.
  • any serovar of S. enterica may be used as the bacterial host for a live vaccine composition for avian influenza, provided the necessary attenuating mutations and antigen-expressing plasmids as described herein are also employed. Accordingly, serovars of S.
  • enterica that may be used in the invention include those selected from the group consisting of Salmonella enterica serovar Typhimurium (“S. typhimurium”), Salmonella montevideo, Salmonella enterica serovar Typhi (“ S. typhi ”), Salmonella enterica serovar Paratyphi B (“ S. paratyphi B”), Salmonella enterica serovar Paratyphi C (“ S. paratyphi C”), Salmonella enterica serovar Hadar (“S. hadar”), Salmonella enterica serovar Enteriditis (“S. enteriditis”), Salmonella enterica serovar Kentucky (“S. kentucky”), Salmonella enterica serovar Infantis (“S.
  • S. typhimurium Salmonella enterica serovar Typhimurium
  • Salmonella montevideo Salmonella enterica serovar Typhi
  • S. typhi B Salmonella enterica serovar Paratyphi B
  • Salmonella enterica serovar Paratyphi C Salmonella enterica serov
  • Salmonella enterica serovar Pullorum (“S. pullorum”), Salmonella enterica serovar Gallinarum (“ S. gallinarum ”), Salmonella enterica serovar Muenchen (“S. muenchen”), Salmonella enterica serovar Anaturn (“S. anatum”), Salmonella enterica serovar Dublin (“S. dublin”), Salmonella enterica serovar Derby (“S. derby”), Salmonella enterica serovar Choleraesuis var. kunzendorf (“S. cholerae kunzendorf’), and Salmonella enterica serovar minnesota (S. minnesota).
  • the vaccine compositions described herein may be administered orally to an individual in any form that permits the Salmonella bacterial strain of the composition to remain alive and to persist in the gut for a time sufficient to elicit an immune response to one or more avian influenza antigens of avian influenza virus and highly pathogenic derivatives expressed in the Salmonella strain.
  • the live bacterial strains described herein may be administered in relatively simple buffer or saline solutions at physiologically acceptable pH and ion content.
  • physiologically acceptable is meant whatever is compatible with the normal functioning physiology of an individual who is to receive a live vaccine composition described herein.
  • bacterial strains described herein are suspended in otherwise sterile solutions of bicarbonate buffers, phosphate buffered saline (PBS), or physiological saline, that can be easily swallowed by most individuals.
  • PBS phosphate buffered saline
  • “oral” routes of administration may include not only swallowing from the mouth a liquid suspension or solid form comprising a live bacterial strain described herein, but also administration of a suspension of a bacterial strain through a nasojejunal or gastrostorny tube, and rectal administration, e.g., by using a suppository comprising a live bacterial strain described herein to establish an infection by such bacterial strain in the lower intestinal tract of the alimentary canal.
  • any of a variety of alternative modes and means may be employed to administer a vaccine composition described herein to the alimentary canal of an individual if the individual cannot swallow from the mouth.
  • the bacteria have genetic modifications which result in the expression of at least one hemagglutinin and one neuraminidase, where each gene is optimized for bacterial expression in at least one codon.
  • the hemagglutinin and neuraminidase genes are further modified to be secreted by the bacteria as heterologous fusion proteins.
  • the neuraminidase and hemagglutinin heterologous fusion proteins are integrated into the chromosome in delta IS200 sites.
  • the bacterial strains are genetically stabilized by deletion of IS200 elements, which reduces their genetic recombination potential.
  • the bacterial strains are genetically stabilized by deletion of phage and prophage elements, which reduces their genetic recombination and transduction potential.
  • the bacterial strains are genetically isolated from phage infection by constitutive expression of the P22 C2 repressor, which reduces their ability to be infected by phage and the subsequent transduction of genes by such phage.
  • the bacterial strains have genetically defined flagellar antigens, or no flagellar antigens, which reduces the immune system elimination of the vector, enhancing its immunization potential in second immunizations.
  • the genetically modified bacteria are used in animals, including humans, birds, dogs and pigs, for protection against avian influenza and highly pathogenic derivatives.
  • kits allows for rapid construction of a bacterial vaccine which is closely matched to an emerging avian influenza or its highly pathogenic derivative.
  • FIG. 1 shows a modified ptrc99a plasmid.
  • the SphI site within the multiple cloning site has been deleted making the upstream SphI site unique and useful for subcloning into pCVD vectors.
  • NotI and PacI sites are added downstream of the t 1 t 2 terminators also for use in subcloning into pCVD vectors.
  • FIGS. 2A and B show a plasmid vectors capable of expressing the H5 or N1 antigens cytoplasmically.
  • Ptrc refers to a functional trc promoter operably linked to a structural coding sequence for an H5 antigen fusion polypeptide.
  • T1 T2 refers to the T1 and T2 transcriptional terminators of the 5S bacterial ribosomal RNA gene.
  • bla refers to the beta-lactamase gene for ampicillin and carbenicillin resistance. Arrows indicate direction of transcription. See text for details.
  • FIG. 3 shows modified ptrc99a plasmid with unique restriction sites engineered into the coding sequence of the N1 gene for rapid exchange of mutations such as the H274Y.
  • FIG. 4A shows a plasmid vectors expressing an antigen (H5 or N1) in a secreted form as fusions with the hlyA protein. Numbers after names of restriction endonucleases indicate specific restriction sites in the plasmid. “Ptrc” refers to a functional trc promoter operably linked to a structural coding sequence for an antigen fusion polypeptide. “ColE1 ori” refers to the colicin E1 origin of replication. 4B shows the hemolysin secretion HlyB and HlyD proteins in a plasmid vector with a different origin of replication, the “M15ori”, which refers the M15 origin of replication. See text for details.
  • FIG. 5 shows ClyA fusion.
  • a plasmid vector for expression of an antigen (e.g., H5 or N1) as a ClyA fusion is shown.
  • the modified trc99a vector of FIG. 1 is used as a cloning and expression vector for a ClyA:antigen fusion.
  • FIG. 6 shows Autotransporter fusion.
  • a plasmid vector for expression of an antigen (e.g., H5 or N1) as a autotransporter (translocator) fusion is shown.
  • the modified trc99a vector of FIG. 1 is used as a cloning and expression vector for the autotransporter:antigen fusion.
  • S refers to a hydrophobic signal sequence.
  • FIG. 7 shows Colicin E3 (ColE3) fusion.
  • a plasmid vector for expression of an antigen (e.g., H5 or N1) as a colicin E3 fusion is shown.
  • the modified trc99a vector of FIG. 1 is used as a cloning and expression vector for the ColE3:antigen fusion.
  • FIG. 8 shows pCVD knockout constructs.
  • A Selection of 5′ and 3′ DNA segments for constructing a pCVD442 chromosomal integration vector for disrupting chromosomal genes and integration of new genes into the chromosome.
  • the 5′ and 3′ segments may be selected completely within the gene (a), partly within and partly outside (b) or completely outside (c) or any combination of the above so long as in each case there is a gap of at least one nucleotide such that the recombination event results in such gap introduced into the gene as a deletion resulting in inactivation of the gene.
  • a foreign gene is inserted such as in FIG. 8B , then the inserted gene also results in a gene disruption following integration and resolution.
  • B Selection of 5′ and 3′ DNA segments for constructing a pCVD442 chromosomal integration vector for disrupting chromosomal genes and integration of new genes into the chromosome.
  • the 5′ and 3′ segments may be selected completely within
  • a localization vector with 5′ and 3′ sequence flanking a multiple-cloning sites (SphI/NotI) into which an expression cassette containing a gene of interest (e.g., an antigen such as H5 or N1, or another gene of interest such as the P22 phage C2 phage repressor protein).
  • a gene of interest e.g., an antigen such as H5 or N1, or another gene of interest such as the P22 phage C2 phage repressor protein.
  • FIG. 9 shows a cloning sequence, from A) synthetic gene within the expression vector to B) chromosomal localization vector.
  • a synthetic gene is generated using standard molecular techniques, the gene is then cloned into an expression vector and then subcloned into a pCVD vector for chromosomal localization.
  • H274Y refers to the histidine to tyrosine mutation that confers oseltamivir resistance.
  • FIG. 10 shows a determination of IS200 17.7 and 19.9 rearrangement/deletion.
  • the Suwwan deletion is a recombination event between two IS200 elements located at 17.7 and 19.9 Cs. If oligonucleotide primers are generated (P1; forward) to unique sequences before the 17.7 and (P2; reverse) after the 19.9, no PCR product will be generated under standard short PCR conditions (typically 500 bp to 10,000 bp) because the distance between the two points is too long (greater than 20,000 bp). However, following a Suwwan deletion, the two points are in relative close proximity and a PCR product will readily be generated.
  • FIG. 11 shows a method to generate strains capable of undergoing the Suwwan deletion in strains lacking the 17.7 Cs IS200.
  • an IS200 can be introduced in order to generate the potential to undergo such a deletion.
  • a chromosomal localization vector derived from pCVD442 can be generated with an cloning sites (SphI/NotI) into which will accommodate foreign DNA.
  • avian influenza genes can be cloned as a codon-optimized synthetic DNA construct and expressed in bacteria including but not limited to Salmonella .
  • Cloning and expression of the avian influenza genes uses standard molecular techniques (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989) and conventional bacterial expression plasmids such as pTrc99a (Pharmacia-Upjohn). This results in a plasmid-based, cytosolic expression of the antigen.
  • the avian influenza antigens can be further modified for secretion as heterologous fusions.
  • fusions can be with previously described for hlyA, clyA, SPATE autotransporter proteins or a novel composition of a fusion with colicin E3 (colE3).
  • colE3 colicin E3
  • cytosolic and secreted constructs can be further modified by integration into the bacterial chromosome using standard techniques of targeted homologous recombination (Donnenberg and Kaper, 1991) where the bacterial expression cassette is inserted in between the 5′ and 3′ flanking sequences as further described below.
  • Bacterial strains such as Salmonella contain a variety of phage and prophage elements. Activation of such phage elements can result in genetic rearrangements and/or liberate such phage as Gifsy and Fels which are capable of transducing other bacterial strains.
  • Such phage elements are known by DNA sequence of entire genomes. If the genome sequence is unknown, such elements may be determined by low stringency DNA:DNA hybridization.
  • DNA sequences associated with phage and prophage elements are disrupted to improve genetic stability and reduce the potential for transduction. Genetic stability is improved by deletion of IS200 and phage/prophage elements.
  • IS200 and phage/prophage elements on the bacterial chromosome are accomplished using standard techniques of targeted homologous recombination (Donnenberg and Kaper, 1991) where 5′ and 3′ flanking sequences of the deletion target (IS200 or phage/prophage elements) are cloned into the pCVD442 vector.
  • Improvement of genetic stability can be determined by assay of phenotypic or genotypic properties such as spontaneous rearrangement of IS200 elements resulting in chlorate resistance (Murray et al., 2004).
  • the ability to rearrange IS200 elements and cause a spontaneous deletion may be determined by assay of spontaneous chlorate resistant bacterial colonies on LB media containing chlorate. These colonies are then subjected to PCR analysis of the genome, combined with DNA sequencing, which is thus definitive in respect to a particular IS200-based deletion in the 17.7 to 19.9 Cs region (See FIG. 8 ).
  • This and other DNA rearrangements, duplications and deletions are also determined by pulse-field gel electrophoresis which compares the DNA banding pattern of the parent strain (control strain) to strains in which rearrangements are to be determined (test strains).
  • the bacterial strains which vector the avian influenza antigens can be altered to genetically isolate them from phage. Genetic isolation is accomplished by limiting the phage integration through constitutive expression of the P22 phage repressor. When exogenous phage enter the repressor inhibits their integration into the chromosome. Under certain circumstances, the repressor may be proteolyticly cleaved by the RecA protein. This may be circumvented by eliminating the RecA protein cleavage site through site-directed mutagenesis (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989).
  • the P22 repressor is cloned into a bacterial expression vector, such as the trc99a vector and results in constitutive expression.
  • the expression cassette may be further modified to be integrated into the chromosome using standard techniques of targeted homologous recombination (Donnenberg and Kaper, 1991) where the trc99a expression cassette is cloned between the 5′ and 3′ flanking sequences of a deletion target (e.g., IS200 or phage/prophage elements) within a pCVD442 vector. Genetic isolation is tested by experimental infection with phage to which the bacteria are normally susceptible.
  • a deletion target e.g., IS200 or phage/prophage elements
  • a genetically isolated strain is recognized by substantially lower infection rates (e.g., 10 fold lower or more) compared to the parent strain, where infection rates are determined by plaque forming units (PFU) of phage, such as P22 phage.
  • PFU plaque forming units
  • the transduction potential of such bacteria is also assayed using standard techniques know to those skilled in the arts, such as the comparison of transducing potential for a metabolic gene (e.g., purl) from the parent strain compared to the modified strain to an identical recipient strain deficient in the same metabolic gene (e.g., delta purl).
  • the genetically isolated strains shows substantially lower (e.g., 10 fold lower or more) ability to have a representative gene transduced to another strain compared to the parent strain.
  • the bacterial strains have genetically defined flagellar antigens, or no flagellar antigens, which reduces the immune system elimination of the vector, enhancing its immunization potential in second immunizations.
  • Strains with defined flagellar antigens are constructed by first selecting substrains that express either the fH1 or fH2 antigens which the bacteria spontaneously generate by inversion of a portion of the gene mediated by the hin recombinase. To select strains expressing either fH1 or fH2, the bacteria are plated to standard growth media and subjected to a colony lift using nitrocellulose or equivalent membrane binding matrix, followed by lysis and blocking of the membrane.
  • fH1 and fH2 are selected using fH1 and fH2 antibodies. The corresponding clone is then purified. These clones are further subjected to deletion of the hin recombinase gene using standard homologous recombination techniques including lamda red recombinase or pCVD vectors specific for disrupting hin, thus fixing their flagellar antigen expression. Furthermore, strains without any flagellar antigens may be constructed by deletion of the fliBC genes using standard homologous recombination.
  • fH1, fH2 or no flagellar antigens have reduced elimination by the immune system when they are used for second immunizations where the first immunization is a bacterial strain with a different flagellar antigen or no flagellar antigen or where the first immunization is a non-bacterial vaccine including an egg-based vaccine.
  • the bacterial strains which vector the H and N antigens of avian influenza and highly pathogenic derivatives are useful as vaccines, resulting protection against infection by influenza strains.
  • a kit according to one embodiment of the invention comprises 1) a bacterial strain, 2) pTrc99a expression vectors containing A) neuraminidase and B) hemagglutinin antigens with unique restriction endonuclease enzymes within the sequence which allows rapid exchange of small segments (such as the N1 amino acid 274) and 3) multiple unique chromosomal localization vectors targeting a variety of genes including IS200s, phage elements (especially Gifsy and Fels) and metabolic genes (such as purl, AroA, etc) for insertion of the pTrc99a expression cassettes with the modified H and N antigens.
  • Bacterial strains useful in the invention include strains of known safety when administered to humans including but not limited to Ty21a, CMV906, CMV908, CMV906-htr, CMV908-htr, Ty800, holavax, M01ZH09, VNP20009. These strains contain defined mutations within specific serotypes of bacteria. The invention also includes the use of these same mutational combinations contained within alternate serotypes or strains. Each of the mutations can be generated by chromosomal deletion techniques known to those skilled in the arts. Generally, the mutational combination includes at least two mutations. Such mutations are made sequentially and generally involve the elimination of antibiotic resistance markers.
  • the process therefore consists of a first step in selection of an appropriate serotype based upon the known species specificity (e.g, S. typhi is human specific and S. typhimurium has broad species specificity including humans, birds, pigs and many other vertebrates).
  • S. typhi is human specific and S. typhimurium has broad species specificity including humans, birds, pigs and many other vertebrates.
  • S. typhi would be appropriate.
  • S. motevidio which have non-overlapping O-antigen presentation (e.g., S. typhimurium is 0-1,4,5,12 and S.
  • S. montevideo is 0-6, 7
  • S. S. typhimurium is a suitable serotype for a prime/boost strategy where S. typhimurium is either the primary vaccine, or the booster vaccine where the primary vaccine is another serotype such as S. typhi or S. montivideo.
  • S. typhimurium is suitable for humans, pigs or birds.
  • a second step follows serotype selection where the first genetic mutation is introduced which may involve the use of antibiotic resistance markers and where any antibiotic resistance makers are then eliminated, followed by a third step where a second genetic mutation is introduced which may involve the use of antibiotic resistance markers and where any antibiotic resistance makers are then also eliminated.
  • Reiteration of genetic deletion and antibiotic marker elimination can be used to supply additional mutations.
  • Methods for reiterative chromosomal deletion and elimination of antibiotic resistance markers are known to those skilled in the arts, including TN10 transposon deletion followed by Bochner selection for elimination of the tetracycline antibiotic resistance marker, lamda red recombinase deletion followed by flip recombinase elimination of the antibiotic resistance marker, and suicide vectors such as those containing sucrasgene (e.g., pCVD442, Donnenberg and Kaper, 1991).
  • the pCVD442 vector is used in the following manner to create specific genetic deletions. First, the desired bacterial serotype is selected, such as Salmonella typhimurium .
  • the desired genetic background to be utilized is selected, such as AroA-, AroD-, htrA- which has been shown to be a safe mutational combination.
  • the genes are then deleted in sequence using the pCVD442 vector as described by Donnenberg and Kaper 1991.
  • the construction of the deletion vector uses DNA sequence for the gene of interest and/or the flanking 5′ and 3′ DNA. Such DNA may be known and previously deposited in a database, or new sequence obtained by methods known to those skilled in the arts such as low stringency hybridization.
  • the isolation genes such as AroA, AroD, htrA or any other known attenuating mutation from Salmonella serotypes where the DNA sequence is not known is accomplished by low-stringency DNA/DNA hybridization of a Salmonella genomic DNA library carried in either E. coli or Salmonella LT2 5010 (e.g., Sambrook et al., 1989 Molecular Cloning: A laboratory manual (2 d Ed.), Cold Spring Harbor Laboratory Press; Low et al., 1999 Nature Biotechnology).
  • a probe for the desired gene such as AroA, AroD, htrA or any other known attenuating mutation is generated from a known homologous gene and its corresponding DNA sequence of such as AroA, AroD, htrA or any other known attenuating mutation respectively, by PCR.
  • This fragment is labeled using 32 P-dCTP and used to probe the Salmonella library at low-stringency conditions consisting of 6 ⁇ sodium chloride/sodium citrate (SSC), 0.1% sodium dodecylsulfate (SDS), 2 ⁇ Denhardts, 0.5% non-fat dry milk overnight at 55° C.
  • SSC sodium chloride/sodium citrate
  • SDS sodium dodecylsulfate
  • 2 ⁇ Denhardts 0.5% non-fat dry milk overnight at 55° C.
  • Flanking DNA representing 5′ and 3′ regions is then cloned into the sucrase vector using standard techniques such that the unification of these regions represents a genetic deletion within the desired gene of at least one nucleotide. Preferably, most or all of entire gene is deleted (See FIG. 8 ).
  • the vector is transformed to the desired strain and selected for antibiotic (ampicillin) resistance.
  • the ampicillin resistance is then eliminated by selection of deletion of the sucrase gene by plating the bacteria to agar plates containing sucrase as described by Donnenberg and Kaper, 1991. Reiteration of these steps targeted at additional genes results in multiple mutations within the desired genetic background.
  • Strains useful in the invention also include novel combinations of mutations including phoP, phoQ, cdt, cya, crp, poxA, rpoS, htrA, nuoG, pmi, pabA, pts, damA, purA, purB, purl, zwf, aroA, aroC, aroD, gua, cadA, rfc, rjb, rfa, ompR, msbB and the Suwwan deletion. Novel combinations are selected by experimental analysis of two factors, attenuation and immunogenicity.
  • LD50 by administration in normal immunocompetent mice is greater than 10 5 , but not more than 10 9 , and/or the LD50 by IV injection is more than 10 4 but not more than 10 8 is desirable, since this is expected to translate into a dose in humans which will neither be too potent and prone to potential overdosing and/or side effects, nor over-attenuated which would result in use of very large doses necessitating vastly greater manufacturing capability.
  • a safe dose (LD 0 ) is first determined in mice, and may be extrapolated to other species on a per weight basis or on a basis of surface area (e.g., meters2).
  • a safe dose is the non-lethal dose determined by a toxicity study using standard methods (Welkos and O'Brian Taylor et al., Proc. Natl. Acad. Sci. USA 84: 2833-2837).
  • a dose 1:100 or 1:1000 of the LD 0 may first be tested and then escalated to a maximum tolerated dose (MTD) defined as the maximum dose having acceptable toxicities which are not life threatening.
  • MTD maximum tolerated dose
  • Immunogenicity is determined by methods know to those skilled in the art including wild type strain challenge and/or analysis for immune response to specific antigens, e.g., ELISA for LPS (e.g., FLOCKTYPE® Labor Diagnostik, Leipzig, Germany) or to the genetically engineered antigens as described further in examples 2.15 and 2.16. Strains which fall into the attenuation range and have the comparatively highest immune response as determined by ELISA and wild type immune challenge using methods known to those skilled in the arts are preferred. By way of example, the following three combinations are generated 1) aroA and purl, 2) aroA and Suwwan, and 3) aroA, purl and Suwwan are generated. DNA sequences for aroA and purl are known for S.
  • the Suwwan deletion is described by Murray et al., 2004.
  • the Suwwan deletion is selected for in ATCC strain 14028 using agar plates containing chlorate. Approximately one in three resistant stains contain the Suwwan deletion, which is confirmed by PCR using primers outside of the two IS200 elements ( FIG. 8 ).
  • the introduction of the Suwwan deletion is not followed by restoring antibiotic sensitivity, since chlorate is not clinically relevant and there is no antibiotic resistance gene inserted in the process.
  • mice are first administered the individual bacterial strain orally at a safe dose (i.e., an LD 0 or less than the LD 10 as defined from the same LD 50 experiment previously performed).
  • a safe dose i.e., an LD 0 or less than the LD 10 as defined from the same LD 50 experiment previously performed.
  • Sub-lethal doses of the attenuated strains are expected to immunize the mice against the lethal wild type strain.
  • a suitable time period for example, 2 to 6 weeks, 1 to 12 weeks, or 1 to 53 weeks
  • either a booster dose also less than the LD 10 may be administered and staged for two additional weeks, or the challenge experiment may be performed.
  • the challenge is performed in the form of an oral administration of a lethal dose of the wild type, usually 10 colony forming units (CFU) or greater, and a survival is monitored over time.
  • CFU colony forming units
  • Strains with the greatest immunization potential result in immunized mice with the longest survival.
  • Immunization can also be determined by immune response to Salmonella antigens, such as the O-antigens, H-antigens or LPS.
  • a determination of anti-LPS is performed using a commercially available ELISA kit. Bacterial strains with the appropriate attenuation and highest level of demonstrated immunization are used for vaccine carriers.
  • the method for selection of the Suwwan deletion has been described by Murray et al., 2004 for the Salmonella typhimurium strain ATCC 14028. Since other Salmonella strains lack the additional IS200 element at Cs. 17.7, they do not undergo this specific chromosomal deletion.
  • the invention further provides a method to allow the Suwwan deletion to occur in other Salmonella strains, by using a sucrase deletion construct as described above which contains the 3′ and 5′ flanking regions which occur in other strains, isolated using analogous primers and providing a multiple cloning site.
  • the IS200 Cs 17.7 is then cloned by PCR into the multiple cloning site of the sucrase vector containing the flanking sequence of the empty IS200 site. Subsequent homologous recombination results in the addition of the IS200 to the site where it was previously absent. Subsequent selection for the Suwwan deletion is then performed, resulting in a strain with the analogous Cs 17 Cs 19 deletion.
  • Codon optimized genes generated by reverse translation (a.k.a., back-translation) of the avian influenza genes or their highly pathogenic derivatives using Salmonella optimized codons and the synthetic gene constructed by annealing overlapping plus and minus strand oligonucleotides.
  • a second codon GCT is added following the ATG start site, the two codons together with an upstream CC constitute the restriction endonuclease site NcoI (CCATGG).
  • the restriction endonuclease site HindIII has been added, thus, a nucleic acid containing this sequence can be restriction digested with NcoI and HindIII and cloned into the NcoI/HindIII sites of the bacterial expression plasmid trc99a (Pharmacia/Upjohn).
  • the trc99a vector is modified to remove the sphI and pstI sites and addition of NotI and PacI sites ( FIG. 1 ).
  • Clones may be further confirmed by restriction endonuclease analysis or DNA sequencing.
  • the NotI and PacI sites are added by inverse PCR, where the primers consist of INVNOTF1
  • the forward primer introduces the NotI and PacI sites, and the reverse primer provides a second NotI site.
  • the linear PCR product is then restriction digested with NotI and self-ligated, and transformed to E. coli . Confirmation of the correct clones is obtained by restriction analysis, where the isolated plasmids now contain NotI and PacI sites or by DNA sequencing.
  • Bacterial expression is tested by any applicable technique known to those skilled in the arts such as ELISA or immunoblot.
  • plasmid can be transferred to a suitable Salmonella strain by standard transformation techniques to comprise a Salmonella strain which expresses the H5 antigen cytoplasmically and is capable of eliciting an immune response.
  • a codon optimized sequence is generated by reverse or back translation, i.e., the conversion of the amino acid sequence into the appropriate DNA sequence. Because of redundancy of the genetic code, many amino acids have more than one possible codon set which will translate to the appropriate amino acid. Recognition sequences representations use the standard abbreviations (Eur. J. Biochem. 150:1-5, 1985) to represent ambiguity.
  • the H5 hemagglutinin gene has a number of known sequence, see e.g., Genbank LOCUS NC — 007362, isolated from a goose in Guangdong, China in 1996, or a more preferably, a recent isolate such as CY019432, obtained from a 26 year old female human infected with avian influenza in Indonesia in 2006, expressly incorporated herein by reference.
  • the sequence begins with four spacer codons for restriction digestion and cloning.
  • Genbank sequence had a second codon inserted (GCT), which is a strong translational second codon in gram negative bacteria.
  • GCT second codon inserted
  • the initiating codon ATG is underlined as well as the stop codon TGA which is followed by the nucleotides for the restriction site HindIII and four spacer codons.
  • Codon optimized N1 orf (SEQ ID NO: 005) is generated by reverse translation of the avian influenza gene using Salmonella optimized codons and the synthetic gene constructed by annealing overlapping plus and minus strand oligonucleotides as described in the example above.
  • a second codon GCT encoding alanine is added following the ATG start site encoding the initiating methionine, the two codons together with an upstream CC constitute the restriction endonuclease site NcoI. Further upstream the nucleotides GACT are added to increase the distance of the restriction site from the end, enhancing the abiligy of the enzyme to cut close to the end.
  • a nucleic acid containing this sequence can be restriction digested with NcoI and HindIII and cloned into the NcoI/HindIII sites of the bacterial expression plasmid trc99a (Pharmacia/Upjohn).
  • Bacterial expression is tested by any applicable technique known to those skilled in the arts such as ELISA or immunoblot.
  • plasmids can be transferred to a suitable Salmonella strain by standard transformation techniques to comprise a Salmonella strain which expresses the H5 antigen cytoplasmically and when administered to an animal is capable of eliciting an immune response as described in example 7.15.
  • the N1 neuraminidase gene has a known sequence, see Genbank LOCUS NC — 007361, expressly incorporated herein by reference.
  • the sequence is further optimized for bacterial expression by addition of the appropriate restriction sites for cloning.
  • An NcoI site is engineered using the start codon together with second codon GCT and a stop codon is added after the final amino acid codon together with an engineered HindIII site and end spacer.
  • Such a synthetically derived DNA sequence can then be cloned into the NcoI/HindIII sites of the bacterial expression plasmid pTrc99a and transformed into the Salmonella strain to result in a vaccine strain expressing the viral antigen.
  • Oseltamivir-resistant neuraminidase is an example of an antigen with an altered amino acid sequence which could change antigenicity.
  • the above synthetic construct in Example 2.5 above which contains restriction sites is further modified, where the synthetic sequence contains mutations representing resistance to osetlamivir, such as the histadine to tyrosine mutation at amino acid position 274 (H274Y).
  • First the trc99a N1 expression construct is restriction endonuclease digested with appropriate sequences.
  • a synthetic DNA construct containing the N1 sequence bearing the H274Y variation is obtained through synthetic construction and ligated into the restriction endonuclease target sites of the previously prepared gene.
  • the plasmid is transfected into a suitable bacterial vector.
  • the new construct is more rapidly generated and when expressed in the bacterial vector, results in a vaccine antigenically matched to the emerging oseltamivir resistant strain.
  • Avian influenza antigen polypeptides expressed from antigen-expressing plasmids or chromosomal constructs in the vaccine strains described herein need not be linked to a signal peptide or other peptide for membrane localization or secretion across the cell membrane.
  • a nucleotide sequence that encodes an H5-HlyA fusion polypeptide useful in the invention is known in the art, and the corresponding encoded H5-HlyA fusion polypeptide has the corresponding amino acid sequence.
  • the antigen-expressing plasmids useful in the invention may be engineered to express an avian influenza antigen polypeptide intracellularly in a host Salmonella strain.
  • antigen-expressing plasmids or chromosomal expression constructs useful in the invention are engineered to express secreted forms of avian influenza antigen polypeptide extracellularly.
  • avian influenza antigen polypeptides expressed from antigen-expressing plasmids in the vaccine strains described herein are preferably linked to a signal peptide or other peptide for membrane localization or secretion across the cell membrane.
  • HlyA hemolysin A
  • H5 nucleotide sequence to result in an hlyA secreted fusion peptide.
  • HlyA fusions are generated using plasmids that provide the 60 C terminal amino acids of HLYA [(Gentschev, et al., 1994. Synthesis and secretion of bacterial antigens by attenuated Salmonella via the Escherichia coli hemolysin secretion system. Behring Inst. Mitt. 95:57-66; Holland et al. U.S. Pat. No.
  • the fusion may also be generated as a completely synthetic DNA construct as described for the hemagglutinin and neuraminidase genes.
  • the sequence begins with four spacer codons for restriction digestion and cloning.
  • Genbank sequence had a second codon inserted (GCT) in the H5 gene, which is a strong translational second codon in gram negative bacteria.
  • the initiating codon ATG is underlined.
  • a SmaI restriction endonuclease site has been added in place of the H5 stop codon to facilitate cloning and the fusion of the peptides, followed by in-frame coding sequence for the 60 C-terminal amino acids of the HlyA gene, which ends with the stop codon TAA (underlined) which is followed by the nucleotides for the restriction site HindIII and four spacer codons.
  • a naturally occurring PacI restriction endonuclease site occurring within HlyA has been conservatively altered to facilitate the potential use of PacI as a restriction site outside of the coding sequence.
  • the secretion of the hlyA fusion requires the presence of the HlyBD gene products.
  • a plasmid containing the genes may be used ( FIG. 4 ), or preferably, the HlyBD genes are cloned within a sucrase vector such as an IS200 phage recombinase, flagellar, or hin pCVD deletion vector.
  • the entire export cassette can be excised from pVDL9.3 as a NotI-digested fragment and cloned into the NotI site of a sucrase vector, which when recombined with the chromosome, results in deletion of the IS200 phage recombinase, flagellar, or hin and insertion of the HlyBD genes into the chromosome.
  • Autotransporter fusions with hemagglutinin and neuraminidase antigens are capable of self-transportation/secretion outside the bacterial cell.
  • Hemagglutinin and neuraminidase fusions with the IgA protease autotranported protein of Nisseria gonorrhoeae are constructed according to the methods of Veiga et al., 2003 J. Virol. 2003 77: 13396-13398) and Oomen et al., 2004 EMBO Journal 23: 1257-1266.
  • the resulting fusion construct when transfected into a bacterial vector, results in a vaccine strain which secretes the neuraminidase and hemagglutinin antigens.
  • Colicin E3 is a bacterial ribosomal RNA inactivating toxin. ColE3 is neutralized within the cells that express it by an antitoxin which inhibits is anti-ribosomal activity.
  • An inactivated ColE3 is cloned from a colE3 containing bacterial strain (e.g., ColE3-CA38).
  • PCR primers consist of a forward primer which clones the start codon with a second added codon and providing an NcoI cloning site and a reverse primer which contains a SmaI (blunt end) cloning site.
  • the PCR primer is situated sufficiently far down the sequence, such that the C-terminal portion of the protein is absent, thus inactivating the toxic activity while retaining the secretion activity.
  • the hemagglutinin and neuraminidase antigens are cut with NcoI and HindIII, blunt end polished and ligated in-frame into the SmaI site of the truncated ColE3 protein. The DNA orientation is then confirmed by restriction analysis and DNA sequencing. When transformed into the bacterial vector, the DNA construct results in secreted hemagglutinin or neuraminidase antigens.
  • IS200 elements can be deleted.
  • Such elements in the Salmonella typhimurium strain LT2 includes LOCUS NC — 003197, having a sequence well known in the art.
  • the IS200 elements contain a transposase with a well known amino acid sequence.
  • Additional IS200 elements can be isolated by low stringency hybridization.
  • the isolation IS200 elements from Salmonella by low-stringency DNA/DNA hybridization of a Salmonella genomic DNA library carried in Salmonella LT2 5010 e.g., Low et al., 1999 Nature Biotechnology.
  • a probe for IS200 is generated from a known IS200 element by PCR. This fragment is labeled using 32 P-dCTP and used to probe the Salmonella library at low-stringency conditions consisting of 6 ⁇ sodium chloride/sodium citrate (SSC), 0.1% sodium dodecylsulfate (SDS), 2 ⁇ Denhardts, 0.5% non-fat dry milk overnight at 55° C. Strongly hybridizing colonies are purified, and plasmids extracted and subjected DNA sequencing. DNA sequence flanking novel IS200 elements is used to generate the 5′ and 3′ regions of a sucrase vector which can then be used to specifically delete that IS200 element.
  • the IS200 located in 17.7 Cs. can be deleted using a 5′ section generated using the PCR primers 2415F1 (IS200 5° F. with SacI)
  • the 5′ section is cloned into the pCVD442 vector using SacI and SphI, and subsequently, after isolation and identification of the appropriate clone, the 3′ section is added using the restriction endonuclease enzymes SphI and SalI.
  • the primers also provide a multiple cloning site containing NotI, PacI, BstY1, SphI, SfiI, Swa1, which can be used to deliver exogenous genes such as the H5 and N1, the lamda repressor C1, or the hlyBD (protein channel) described further below.
  • Bacterial strains containing phage or prophage elements may have the phage enter a lytic cycle in which they may undergo recombination inversion.
  • Bacterial strains such as Salmonella contain Fels and Gifsy prophage.
  • the Fels prophage recombinase/invertases can be deleted using the pCVD442 homologous recombination system as described above for IS200 elements. Deletion results in the inability to excise the phage DNA and therefore is unable to undergo the lytic cycle or genetic recombination.
  • the Fels-1 invertase has a well known amino acid and DNA sequence.
  • the Fels-2 recombinase/invertases also have known amino acid sequences, and DNA sequences.
  • Vertibrate animals including mice, birds, dogs, cats, horses, pigs or humans are selected for not having any known current or recent (within 1 year) influenza infection or vaccination. Said animals are pre-bled to determine background binding to, for example, H5 and N1 antigens.
  • the Salmonella expressing H5 and N1 are cultured on LB agar overnight at 37°. Bacteria expressing other H and or N antigens may also be used.
  • a pharmacologically suitable buffer such as PBS and they are diluted to a concentration of 104 to 10 9 c.f.u./ml in a pharmacologically suitable buffer on ice, warmed to room temperature and administered orally or intranasally in a volume appropriate for the size of the animal in question, for example 50 ⁇ l for a mouse or 10 to 100 ml for a human.
  • the actual dose measured in total cfu is determined by the safe dose as described elsewhere in this application.
  • a booster dose may be given.
  • the booster may be the same as the initial administration, a different species, a different serotype, or a different flagellar antigen (H1 or H2) or no flagellar antigen.
  • an additional blood sample may be taken for further comparison with the pretreatment and 2 week post treatment.
  • the host bacterium (the bacterium the chromosome of which is engineered to encode a heterologous antigen) can be E. coli or any other enteric bacterium, including Salmonella, Bordetella, Shigella, Yersenia, Citrobacter, Enterobacter, Klebsiella, Morganella, Proteus, Providencia, Serratia, Plesiomonas, and Aeromonas , all of which are known or believed to similar to the promoters of E. coli and Salmonella .
  • enteric bacterium including Salmonella, Bordetella, Shigella, Yersenia, Citrobacter, Enterobacter, Klebsiella, Morganella, Proteus, Providencia, Serratia, Plesiomonas, and Aeromonas , all of which are known or believed to similar to the promoters of E. coli and Salmonella .
  • BCG Bacille Calmette-Guerin
  • the promoter used can be native to the species of the host bacterium, or can be a heterologous promoter (i.e., from a species other than that of the host bacterium) engineered into the host bacterium along with the heterologous antigen coding sequence, using standard genetic engineering techniques. Multiple heterologous antigen coding sequences linked to the same or different promoter sequences can be inserted into a given chromosome, using techniques analogous to those set forth above, to produce a multivalent vaccine strain.

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US11/859,569 US20080124355A1 (en) 2006-09-22 2007-09-21 Live bacterial vaccines for viral infection prophylaxis or treatment
AU2007300519A AU2007300519A1 (en) 2006-09-22 2007-09-24 Live bacterial vaccines for viral infection prophylaxis or treatment
CN200780043473A CN101720228A (zh) 2006-09-22 2007-09-24 用于预防或治疗病毒感染的活菌疫苗
EP07838725A EP2081593A4 (fr) 2006-09-22 2007-09-24 Vaccins bacteriens vivants pour la prophylaxie ou le traitement d'une infection virale
RU2009115177/15A RU2009115177A (ru) 2006-09-22 2007-09-24 Живые бактериальные вакцины для профилактики и лечения вирусной инфекции
PCT/US2007/020578 WO2008039408A2 (fr) 2006-09-22 2007-09-24 Vaccins bactériens vivants pour la prophylaxie ou le traitement d'une infection virale
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US13/369,333 US8440207B2 (en) 2006-09-22 2012-02-09 Live bacterial vaccines for viral infection prophylaxis or treatment
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US8440207B2 (en) 2013-05-14
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US9315817B2 (en) 2016-04-19

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