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WO1998033386A1 - Procedes d'apport d'antigenes permettant d'effectuer des vaccinations avec un vecteur vivant - Google Patents

Procedes d'apport d'antigenes permettant d'effectuer des vaccinations avec un vecteur vivant Download PDF

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WO1998033386A1
WO1998033386A1 PCT/US1998/001780 US9801780W WO9833386A1 WO 1998033386 A1 WO1998033386 A1 WO 1998033386A1 US 9801780 W US9801780 W US 9801780W WO 9833386 A1 WO9833386 A1 WO 9833386A1
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strain
fetus
mutant
protein
sapa
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PCT/US1998/001780
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Martin J. Blaser
Stuart A. Thompson
Joel Dworkin
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Vanderbilt University
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Priority to US09/355,793 priority Critical patent/US6803231B1/en
Priority to AU60503/98A priority patent/AU6050398A/en
Publication of WO1998033386A1 publication Critical patent/WO1998033386A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/105—Delta proteobacteriales, e.g. Lawsonia; Epsilon proteobacteriales, e.g. campylobacter, helicobacter
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/205—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Campylobacter (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0258—Escherichia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0275—Salmonella
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates generally to the fields of immunology and vaccine technology. More specifically, the present invention relates to methods of delivering antigens for vaccination with a live vector.
  • microorganisms can alter their surface properties, allowing a fraction of the population to preadapt to environmental changes, is by varying protein expression through programmed genomic DNA rearrangements ( 1). Phase, antigenic, or size variation of expressed surface proteins are governed by mechanisms including transposition and DNA inversion. During transposition, a silent gene is activated by movement to an expression site where it displaces the currently expressed gene. In DNA inversion, a segment of DNA is cut, inverted, and then rejoined by a site-specific recombinase. The invertible DNA segment may contain either a promoter that directs expression of fixed structural genes or structural genes controlled by a fixed promoter. Transposition and inversion differ in both the enzymes used and in the number of genes that can be controlled (many versus two).
  • Campylobacter fetus a bacterial pathogen of ungulates and humans, is covered by a paracrystalline surface (S-) layer, composed of high molecular weight S-layer proteins (SLP) that masks most of the underlying gram-negative surface features (2). More than 300 bacterial genera have been described that possess S-layers (3).
  • S-layer renders C. fetus cells resistant to serum killing by prohibiting the binding of C3b (4), and the S-layer proteins themselves may change, permitting antigenic variation (5,6).
  • S-layer proteins are encoded by 7-9 tightly clustered and partially homologous promoterless gene cassettes (7,8). Since previous studies show that C. fetus can express alternative S-layer proteins (4-6,9), there is only a single promoter for S-layer proteins expression present on a 6.2 kb invertible element (9), and the structural genes flanking the promoter are subject to substitution (9), both the promoter and the eight structural genes ⁇ sapA and its homologs) may rearrange strictly by inversion.
  • Campylobacter fetus is able to colonize the mucosa of the gastrointestinal and/or genitourinary tracts of mammals, birds and reptiles. Colonization of the wild-type organism lasts for years and can cause disease. Essential for this long term colonization is the ability to produce the S-layer proteins and
  • Campylobacter fetus has the means to change the S-layer proteins and thus the crystal struture and the particular forms of antigenicity. This antigenic variation is required for the persistence of the organism in its environmental niche. Campylobacter fetus accomplishes this antigenic variation by posssessing 7-9 highly homologous gene cassettes, called sapA homologs (sapA , sapA l , sapA2 , etc.) which encode a different S-layer protein. Each of these homologs contains a 5' region of about 600 base pairs which is completely conserved from homolog to homolog and is necessary for binding of the S- layer protein encoded by that homolog to the lipopolysaccharide molecule anchored in the bacterial outer membrane.
  • sapA homologs sapA , sapA l , sapA2 , etc.
  • the remainder of the open reading frame (ORF) is different for each homolog but semi-conserved regions exist.
  • Wild type . fetus strains are able to rearrange their chromosomal DNA so that the sapA homolog positioned downstream of a unique promoter is then expressed. This rearrangement occurs at a frequency of about lO -4 and is recA dependent.
  • RecA is a protein encoded by recA and which is involved in homologous recombination and in repair of breaks of DNA strands.
  • C. fetus S-layer proteins are secreted in the absence of an N-terminal signal sequence.
  • SLP proteins contain a signal sequence located within the C-terminous of the protein and are secreted through a type I protein secretion system encoded by the sapCDEF operon of four overlapping genes.
  • Analysis of the C- termini of four C. fetus SLPs revealed conserved- structures that are potential secretion signals.
  • a C. fetus sapD mutant neither produced nor secreted SLPs.
  • E. coli expressing C. fetus sapA a n d sap CDEF secreted SapA indicating that the sapCDEF genes were sufficient for SLP secretion.
  • the prior art is deficient in the lack of effective means of delivering antigens for vaccination with a live vector.
  • the present invention fulfills this longstanding need and desire in the art.
  • a mutant C. fetus strain in which each of the cassettes is replaced by a heterologous antigen.
  • the s apA homologs are altered by a DNA cassette inserted so that the encoded SLP represents a chimera between the native SLP and the peptide encoded by the cassette.
  • the inserted DNA cassettes retain 3' s apA sequences that encode the C-terminal secretion signal sequences in order to ensure secretion of the chimeric protein.
  • Representative examples of cassettes that can be inserted in this fashion include immunogens related to Salmonella, Campylobacter jejuni, E.
  • this strain can be used to immunize a host to develop mucosal and systemic immune responses to each of the immunogens .
  • a mutant C. fetus strain in which all but one of the cassettes are replaced by a heterologous antigen.
  • one of the cassettes e.g., s apA 2
  • the advantage of this construction is that it also can induce immunity to C. fetus based on the single full-length SLP produced.
  • a mutant C. fetus strain in which recA is mutagenized.
  • the DNA rearrangements permitting s ap A antigenic variation can not occur at any detectable frequency.
  • the C. fetus strain can only produce one of the SLPs encoded by one sapA homolog.
  • This strain can be used, therefore, to colonize the host briefly, i.e., until protective immunity has developed to that homolog. Subsequently, the host immune response eliminates the organism.
  • this mutant would provide effective immunity against subsequent C . fe tu s infection and is useful for vaccination of ungulates (including sheep, cattle and horses) in which infectious abortion and/or infertility can occur after the wild-type infection.
  • a C. fetus strain in which recA is mutagenized and the expressed s a p A homolog is a chimera involving a heterologous peptide .
  • a mutagenized s apA homolog expresses a chimeric protein including a heterologous antigen.
  • the strain is then passaged in vitro so that the chimeric homolog is in the expression position and then a re cA mutation is made.
  • This strain now essentially expresses only the chimeric protein thus providing a means to immunize a host to that antigen.
  • the duration of colonization in the host is brief and this attenuated C. fetus strain allows safe immunization for the selected antigen.
  • a mixed mutant C. fetus strains each including a sap A chimera which is also a recA mutant.
  • mutants are constructed in which a single s apA homolog is mutagenized to encode a different chimeric protein representing a different heterologous antigen.
  • Each mutant is also RecA-deficient due to mutation in recA .
  • a host is inoculated with a mixture of two or more of these strains to provide immunization to the requisite antigens. Each strain is short-lived in the immunized host.
  • a strain of E. coli carrying plasmids encoding the sap CDEF proteins that permits the secretion of chimeric proteins containing the SapA C-terminal secretion signal.
  • the secreted protein is encoded by an altered s ap A homolog that has been engineered so that the 5' section of the homolog-specific region is replaced by a DNA cassette encoding a heterologous antigen. Since the chimeric protein would be secreted by E. coli into the culture medium, this system provides a method for obtaining a large amount of the chimeric protein in a relatively pure form.
  • Figure 1 shows the schematic representation of serial experiments to utilize km - and c m -cassettes to examine S-layer proteins gene rearrangement ( Figure 1A). Introduction of cm into strain 23D by marker rescue led to insertion into sap A to create 23D:AC 100.
  • This strain was selected on chloramphenicol- containing medium, and as expected was chloramphenicol (C) resistant, and was S " (no S layer) and not resistant to serum (S) and kanamycin (K), as previously reported (9).
  • Strain 23D:AC200, expressing saphl was further mutagenized by introduction of km into saphl to create the 23D:ACA2K100 series. These strains were S " and serum- and chloramphenicol- sensitive but kanamycin-resistant.
  • Figure IB shows the immunoblot of C. fetus strain 23D and selected mutants into which cm or c m and k m were inserted into S-layer protein gene cassettes. As expected, all the serum-resistant strains expressed S-layer proteins (of 97 or 127 kDa) recognized by antiserum to conserved C. fetus S-layer proteins determinants whereas there was no expression for strains maintained on either antibiotic.
  • Figure 2 shows the Southern hybridization of Hindi
  • Figure 2A or Pstl (Figure 2B) digestions of chromosomal DNA from C. fetus 23D and ACA2K series mutants using probes to km, cm , the promoter region, the s ⁇ A-specific 3' region, the sap A 1 - middle region, or the s ap A 1 -specific 3' region. Each probe hybridized to a single fragment regardless of the phenotype of the C. fetus strain.
  • Figure 2C shows the mapping of S-layer protein gene cassette arrangement by PCR.
  • PCRs were performed with template chromosomal DNA from strains 23D and ACA2K mutants using s ⁇ A-specific 3' region forward (sap A) and km reverse (k m ) primers (left 4 lanes), sap Al -specific 3' region forward (sap A l ) and km primers (middle 4 lanes), or sapA and s ⁇ A l -specific 3' region reverse (sapAl) primers (right 4 lanes).
  • Figure 2D shows the cumulative restriction maps of the 4 strains presented in Figure 2A-2C. The location of the probes as indicated from the hybridications is shown under the map for each strain.
  • sapA x represents an uncharacterized S-layer protein gene cassette; arrows represent direction of transcription; solid lines represent expressed genes, dashed lines represent silent genes; P over bent arrow represents the sap A promoter; the heavy line represents the 6.2 kb invertible promoter-containing element, flanked by opposing S-layer protein gene cassettes.
  • the asterisks represent the palindromic putative recombinase recognition sites (TTAAGGAaTCCTTAA) present in the 5' conserved region of each
  • S-layer protein gene cassette (7), and restriction sites are indicated: H, Hindi; N, Ndel; P, Pstl.
  • Figure 3 shows the proposed model of molecular events involved in S-layer protein gene cassette rearrangement by DNA inversion.
  • DNA inversion between two oppositely oriented cassettes follows DNA strand exchange at the putative recombinase target site (asterisk) found upstream of each S-layer protein gene cassette within the 5' conserved region (small grey box) (8).
  • Patterned boxes represent variable regions of S-layer proteins gene cassettes.
  • a 6.2kb intervening segment is topologically reversed leading to ordered rearrangement of the S- layer protein gene cassettes.
  • Inversion of DNA segments containing the promoter ('P' over bent arrow) permits expression of alternate S-layer protein gene cassettes (mRNA, arrow).
  • Figure 4 shows a schematic representation of the sapA invertible region, showing the sapCDEF genes, the locations of the divergent sapA and putative sapCDEF promoters (bent arrows), and the clones (pIR15, pIR13, pIR12, and pIR20) from which the invertible region sequence was determined. The hatched areas represent the ca.
  • Figure 5 shows a phylogram generated by parsimony analysis, demonstrating the relatedness of ABC transport proteins from type 1 secretion systems.
  • the percent of amino acid similarity (%Sim) and identity (%ID) with C. fetus SapD is shown at the right.
  • the bold numbers adjacent to the phylogram branches indicate the percentage of 1000 bootstrap replicates supporting the clustering of those branches. Branches without bootstrap values were clustered in less than 50% of bootstrap replicates.
  • Figure 6 shows an immunoblot detected with antiserum to SapA demonstrating the secretion of SapA from E . coli expressing C. fetus sapCEEF.
  • Lanes 1 and 2 One mg of whole cell proteins (lanes 1 and 2), or the amount of TCA-precipitate proteins present in 250 ml of culture supernatant (lanes 3 and 4) were analyzed. Lanes 1 and 3, C600 (pAMPl+pBCGYCl); and lanes 2 and 4, C600
  • Figure 7 shows several models of chimeric sap homologs for the use of C. fetus as a live vector for antigen delivery to the mucosal immune system.
  • the left large gray box represents the N-terminal domain required for binding of the protein to the cell surface
  • the right sided smaller gray box represents the C-terminal domain required for secretion of the protein.
  • the middle region shows the native sap homolog, and then in other examples, antigens related to influenza, HIV, Shigella, a model B-subunit of a toxin, or a model A subunit of a toxin.
  • the protein would be secreted without binding to the C. fetus cell surface.
  • RecA at right refers to possible RecA status of the host C . fetus strain. If the strain is RecA " then it would only express a single cassette on its surface, but if RecA + (wild type), it would be able to switch the chimera it expressed. DETAILED DESCRIPTION OF THE INVENTION
  • S- layer protein surface layer protein
  • km kanamycin-resistance gene
  • cm chloramphenicol-resistance gene
  • NHS normal human serum
  • programmed gene rearrangements are employed by a variety of microorganisms, including viruses, prokaryotes, and simple eukaryotes, to control gene expression.
  • viruses prokaryotes
  • simple eukaryotes simple eukaryotes
  • the mechanism of variation is not thought to involve DNA inversion.
  • Campylobacter fetus a pathogenic gram-negative bacterium, reassorts a single promoter, controlling S-layer (surface) protein expression, and one or more complete open reading frames strictly by DNA inversion. Rearrangements are independent of the distance between sites of inversion.
  • S-layer proteins were secreted from C. fetus via a type I protein secretion system encoded by the s ap CD EF operon.
  • Secretory signal sequences for the SapA family of secreted proteins was localized to the carboxy-terminus of these proteins.
  • the present invention is directed to a mutant C . fetus strain useful for vaccinating an animal to Campy lo bac te r fetus, wherein said strain is mutated to contain a DNA cassette encoding a heterologous protein. In one embodiment of the mutant C.
  • the heterologous protein is a S-layer protein.
  • the encoded S-layer protein represents a chimera between the native S-layer protein and the peptide encoded by the cassette.
  • the cassette is selected from the group consisting of Sa lmon e lla , Shig ella, Campylobacter jejuni, E. coli 0157:H7, human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV) and other animal pathogens.
  • the mutant C is selected from the group consisting of Sa lmon e lla , Shig ella, Campylobacter jejuni, E. coli 0157:H7, human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV) and other animal pathogens.
  • the cassette contains a 5' binding region and 3' secretion signal region and the protein is inserted between said binding region and said signal region.
  • the cassette contains a 3' secretion signal but has no binding region.
  • the protein is an antigen or a therapeutic agent.
  • the present invention is also directed to a method of immunizing a host to develop mucosal and systemic immune responses to an immunogen, comprising the step of administering to said host a pharmacologically effective dose of the strain described herein.
  • the present invention is also directed to a mutant C . fetus strain, wherein recA is mutated so that no functional RecA protein is produced, the DNA rearrangements permitting s apA antigenic variation occur at a very low frequency and wherein said C. fetus strain can only produce one of the S-layer proteins encoded by one sapA homolog.
  • this strain contains a sapA homolog expressing a chimeric protein including a heterologous antigen.
  • the present invention is also directed to a mixture of mutant C. fetus strains, wherein each strain includes a s apA chimera which is also a re cA mutant, wherein a single sap A homolog is mutated to encode a different chimeric protein representing a different heterologous antigen and each mutant is also RecA-deficient due to mutation in r e cA .
  • the present invention is also directed to a method of immunizing a host to develop mucosal and systemic immune responses to an immunogen, comprising the step of administering to said host a pharmacologically effective dose of these strains.
  • the present invention is also directed to a strain of bacteria modified to express SapCDEF genes
  • the strain is
  • a heterologous protein is expressed as a chimeric protein composed of sequences of heterologous origin, sequences that direct the secretion of said chimeric protein to the cell surface and sequences that direct the binding of the secreted chimeric protein to the lipopolysaccharides of the bacterial cell surface via the sapCDEF directed type 1 secretory system.
  • the present invention is also directed to a method of immunizing a host to generate immune responses to an immunogen,. comprising the step of administering to said host a pharmacologically effective dose of this strain.
  • Wild-type S + (possessing an S-layer) C. fetus strain 23D and spontaneous S " (no S-layer present) mutant strain 23B have been extensively characterized (4,5, 12, 13).
  • Other C. fetus strains used were defined mutants derived from strain 23D, as described below. Stock cultures were stored and grown as described elsewhere (8).
  • Chromosomal DNA was prepared from 48 hour plate cultures, as described (9). Plasmids were isolated by the procedure of Birnboim and Doly ( 15). All other standard molecular genetic techniques were done, as described (14).
  • Mutant C. fetus strains were created by mobilization of donor pKO500 or pKO505 plasmid constructs by conjugal mating as described elsewhere (9) with sequential selection (as shown in Figure 1) on media containing 30 ⁇ g/ml kanamycin or 15 ⁇ g/m l chloramphenicol, or in the presence of 10% normal human serum (NHS), as described ( 16).
  • pKO500 suicide hybrid plasmid with the sap A open reading frame disrupted with a chloramphenicol- resistance gene (cm ) (17) located 127 base pairs into the open reading frame.
  • pKO505 suicide hybrid plasmid with the sap A 2 open reading frame disrupted with a promoterless kanamycin- resistance gene (18) located 127 base pairs into the open reading frame .
  • Antiserum to the 97 kDa S-layer proteins of type A strain 82-40LP was raised in adult New Zealand White female rabbits and shows broad recognition of C. fetus S-layer proteins as described (5).
  • To analyze wild-type and transconjugant C. fetus strains for S-layer proteins expression cells were harvested from plates, lysed in sample loading buffer and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting was performed as detailed (5, 13).
  • C. fetus chromosomal DNA was digested with Hindi and processed exactly as described (12).
  • Probes included the gel- purified PCR products specific for the sap A promoter region (9) , km , cm, 3' sapA region ( 1649 to 2760 bp), middle sapA l region (620 to ( 1381 to 2763 bp). Probes were 3 extension with random hexameric oligonucleotides (19). Polymerase chain reactions were performed as described (9).
  • Primers used include: km F, 5'-TGTAGAAAAGAGGAAGGAAA-3' (SEQ ID No: 1); km R; 5'-CTAAA ACAATTCATCCAGTA-3' (SEQ ID No: 2); cm F, 5'-AGTGGATAGATTTATGATATAGTG-3' (SEQ ID No: 3); cm R, 5'-T TTATTTATTCAGCAAGTCTTG-3' (SEQ ID No: 4); sapA F middle, 5'-CATCTCTACAGCAGCAAAAG-3' (SEQ ID No: 5); sapA F 5'-GCGGAGATAATGTTGTAGTTGAT G-3' (SEQ ID No: 6); sap A R, 5'-AACTTTAAGAT CTAGCGTACC-3' (SEQ ID No: 7); sapAl F middle, 5'-AGGGTACTGATTTAGACGATA-3' (SEQ ID No: 8); sapAl F 3', 5'-GCTGGATTTACA
  • Phenotypic variation is associated with nested DNA inversions
  • the promoter region, the sapA -3' region, the sapA l middle region and the sap A l-3' region probes hybridized to 8.5 kb, 4.8 kb, 4.8 kb and 4.3 kb Hindi fragments, respectively, in wild-type strain 23D (Figure 2A). Based on these and previous Southern hybridization and PCR data (9), the location of these genes relative to one another in wild-type strain 23D was defined
  • DNA inversion events occur independent of the distance between recombination sites
  • the frequency of the DNA inversion events was determined, involving promoter alone, or promoter and one or more of the gene cassettes.
  • the mutant strains (TABLE 1 ) provided easily definable phenotypes with which to assess the inversion events . These were measured by conducting experiments in which the strains were examined for resistance to a selection that would be lethal (exposure to kanamycin, chloramphenicol, or serum) unless defined inversions allowing expression of genes to overcome the exposure had occurred. Events involving inversion of the promoter-containing element alone, or together with one or two open reading frames, occurred at nearly equal frequency ( ⁇ 1 to 2.6 X 10 "4 ) (TABLE 1 ).
  • the 6.2 kb invertible region between two s ap A homologs was characterized.
  • the 6.2 kb fragment was first amplified by PCR, and this product was subcloned into pAMPl to yield the plasmid pIRlOO.
  • this subcloned fragment was used as a probe to isolate a series of overlapping plasmid clones derived from a C. fetus 23D genomic library constructed in ⁇ ZAPII.
  • Four of these, designated pIR15 , pIR13, pIR12, and pIR20 represented the majority of the invertible region and were subjected to DNA sequence analysis.
  • a DNA segment was amplified from C. fetus 23D genomic DNA by PCR using appropriate primers and subcloned into pT7Blue. To avoid the problem of PCR-induced errors, three independent subclones were sequenced and in each case the sequence was identical.
  • the DNA sequence of the 6229 bp invertible region from strain 23D was predicted to contain four open reading frames, which were designated sapCDEF ( Figure 4).
  • the sapC gene began 596 bp from the initiation codon of the oppositely-oriented upstream sap A homolog.
  • the sap C open reading frame was 1035 bp in length and was immediately followed by the sapD, sapE, and sapF open reading frames, which were 1752, 1284, and 1302 bp, respectively.
  • Each gene in this cluster had a typical ribosome binding site, and overlapped the preceding gene, by 14 bp (sapC/D), 1 bp (sapD/E), and 11 bp (sapE/F).
  • the sapF gene ended 287 bp upstream of the sapA homolog located downstream.
  • the 74 bp preceding the ATG codons initiating translation of the sap A homologs flanking sapCDEF were identical to each other ( Figure 4).
  • These conserved segments have previously been noted upstream of the three characterized sapA homologs, and may play a role in the inversion of this DNA segment.
  • sequences resembling ⁇ sites were present at positions 31-38 and 6192- 6199.
  • Several potential ⁇ 70 -like promoters were noted 44-243 bp upstream of sap C.
  • putative s ap CDEF promoters were oriented in the opposite direction to the sap A promoter, with the two -35 regions separated by 200-380 bp. Due to the overlapping nature of the s ap CD EF genes and the lack of other putative promoters, it is likely that they are co-transcribed.
  • the s ap C open reading frame predicted a protein product of 344 amino acids (39.7 kDa) that had no significant similarities in the non-redundant database maintained by NCBI.
  • the products of the sapDEF genes had high similarity to proteins encoding type I secretion systems.
  • SapD (584 amino acids, 64.0 kDa) was related to the ABC family of transporters, especially those that are involved in the translocation by type I secretion systems of proteases, lipases, hemolysins, and leukotoxins across the envelopes of gram-negative bacterial pathogens.
  • the degree of similarity between SapD and these proteins was between 68% similarity (with P s e u d o m o n a s a e r u g i n o s a AprD) and 50% similarity (A c t i n o b a c i l l u s actinomycetemcomitans LktB).
  • SapD contained two motifs found in such proteins, an ATP/GTP binding site (GPSAAGKS ; Walker Box A, amino acid residues 365-377) and a peptide (LSGGQRQRVALA, amino acid residues 468-479) that is a signature sequence for ABC transporters.
  • the SapE protein (428 aa, 47.9 kDa) was similar to the MFP proteins of type I transporters, typified by P. aeruginosa AprE (52% similar) and E. coli HlyD (49% similar).
  • SapF (434 amino acids, 49.4 kDa) was related to the outer membrane component of type I systems, such as P. aeruginosa AprF (45% similar), Erwinia chrysanthemi PrtE (41 % similar), and E. coli TolC (47% similar). Parsimony and bootstrap analyses supported the classification of the C. fetus transport proteins on phylogenetic branches distinct from other type I secretion apparatuses (Figure 5).
  • a derivative of type A strain 23D containing an insertional mutation in sapD was constructed.
  • an ap hA cassette was inserted into a unique Bglll site within s apD and transformants were selected on plates containing kanamycin. This new clone was designated pIR131.
  • the sapD insert containing the resistance fragment was subcloned into suicide vector pILL570 to yield pILL131 , which was transformed into E. coli S I 7- 1.
  • Transformants were selected from trimethoprim/kanamycin plates and verification of pILL131 was made by digestion with Hindlll.
  • the mob + E. coli strain S 17- 1 containing IncP DNA transfer functions and pILL 131 was used as the conjugation donor, and C. fetus wild-type strain 23D was the recipient. Approximately five thousand transconjugants were recovered (frequency of 4 x 10 " 6 transconj ugants per recipient) . Chromosomal DNA from one of these strains was extracted and digested with Ndel for Southern analysis, using hybridization probes for sapD, aphA , and pILL570. The sapD probe hybridized with a 4.2 kb Ndel fragment in wild-type strain 23D, exactly as predicted from the DNA sequence of the invertible region.
  • the size of the Ndel fragment in strain 97-205 hybridizing to the sapD probe was 5.6 kb indicating the incorporation of the 1.4 kb ap hA cassette in sapD, as expected.
  • the hybridization of the aphA probe with a fragment of 5.6 kb in 97-205 but not in 23D was consistent with this observation.
  • a pILL570 probe did not hybridize with DNA from 97-205, indicating that 97-205 was derived from a double crossover event in which only the mutagenized sapD allele was incorporated into the chromosome.
  • PCR experiments using s ap D - and ap h A -specific primers confirmed the Southern hybridization data. These results indicate that a strain containing an insertional mutation in sapD has been successfully constructed.
  • each of these peptides was analyzed using the programs of Garnier et al.
  • the predicted C-terminal secondary structures ( ⁇ -helix, ⁇ -sheet, amphipathic peptides, and turn- forming residues) were then superimposed on the alignment of the 4 C-terminal peptides.
  • Three of these peptides (SapA, SapAl , and SapB) consisted of segments predicted to form sheet-sheet- helix-sheet, with each of these domains separated by turn- forming.
  • the C-terminal domain of SapA2 was predicted to form a structure of helix-sheet-helix-sheet.
  • S-layer proteins expression in C. fetus is based on the single sapA promoter present on an invertible 6.2 kb element, that is bracketed by inverted repeats and oppositely oriented cassettes, sapA and sap A2, that are subject to substitution (9).
  • the present invention demonstrates that DNA rearrangement can involve inversion of this element in concert with one or more of the tandemly arrayed S-layer protein gene cassettes.
  • DNA inversion has been believed to involve mutually exclusive promoter or structural gene inversion. Either the promoter inverts relative to fixed structural genes (26,27), or structural genes invert downstream of a fixed promoter (28-30) permitting expression of two alternate gene copies ( 10, 1 1).
  • the system of DNA inversion in C. fetus is novel because it combines the features of each mechanism as both the promoter and the structural genes are mobile, which permits the shuffling of complete genes and their ultimate expression ( Figure 3). Rearrangement of the S-layer protein gene cassettes permits the organism to vary S-layer protein expression and surface antigenicity allowing for evasion of host immune responses. This inversion system differs from the Mycoplasma pulmonis vsa gene inversion, which rearranges only incomplete coding regions and demonstrates less sequence stability.
  • the present invention further indicates that inversion occurs randomly between open reading frames of opposite orientation independent of the size of the intervening DNA segment.
  • the economy of a simple inversion system may be especially useful for C. fetus which has a relatively small ( 1.1 megabase) genome (32); the strict conservation of both coding and non-coding regions related to the sap homologs in type A and type
  • B strains (7) is consistent with the importance of this efficient system.
  • the present invention expands the paradigms of DNA rearrangement, in which large gene families of complete open reading frames can reassort by inversion so as to vary the surface protein expression of the microbe.
  • cassettes that can be inserted are immunogens related to Salmon e lla, Campylobacte r jejuni, E. coli 0157:H7, human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV) as well as venereal, gastrointestinal, or respiratory pathogens of humans, cattle, sheep, poultry, horses, swine, and reptiles.
  • the replacement is constructed so that the encoded S- layer protein is a tripartite chimera consisting of the N-terminal LPS-bining domain of the S-layer protein, a central region composed of the heterologous antigen, and a C-terminal segment containing the S-layer protein secretion signal.
  • This protein would be able to be secreted from the cell by means of the C-terminal secretion signal and into the LPS on the C. fetus cell surface, thereby exposing the heterologous antigen to the host immune system.
  • the natural cassettes are replaced by the chimerae, one embodiment of the invention is the expectation that carriage of this organism is substantially shorter than for the wild type strain and thus, self-limiting. This strain should colonize for less than 3 months allowing the host to develop a mucosal and systemic immune response to each of the immunogens.
  • one may use a mutant C . fetus strain in which all but one of the cassettes is replaced by a heterologous antigen.
  • This embodiment is similar to Example 12 except that one of the cassettes (e.g., sapA2 ) remains in its native configuration but that the others are mutagenized to form chimerae.
  • the advantage of this construction is that one can induce immunity to C. fetus based on the single full-length S- Layer protein produced.
  • the C . fetus strain in which recA is mutagenized
  • the C . fetus strain can only produce one of the S-Layer proteins encoded by one sapA homolog. This strain should only colonize the host briefly, i.e., until protective immunity has developed to that homolog. Subsequently, the host immune response should eliminate the organism.
  • this mutant would provide effective immunity against subsequent C. fetus infection and is useful for vaccination of ungulates (including sheep, cattle and horses) in which infectious abortion and/or infertility can occur after the wild-type infection.
  • EXAMPLE 21 C. fetus strain in which r e c A is mutagenized and the sapA homolog that is being expressed is a chimera involving a heterologous peptide
  • a sapA homolog is mutagenized to allow expression of a chimeric protein including a heterologous antigen.
  • the strain is then passaged in vitro so that the chimeric homolog is in the expression position and then a recA mutation is made.
  • This strain now expresses only the chimeric protein which can serve as a means to immunize a host to that antigen. Since the strain can not vary and is not producing a native S-Layer protein, the duration of colonization in the host is brief (days). This attenuation of the C. fetus strain allows safe immunization for the selected antigen.
  • Each mutant is also RecA-deficient due to mutation in recA .
  • a host is inoculated with a mixture of two or more of these strains to provide immunization to the requisite antigens.
  • Each strain is short-lived in the immunized host.
  • Each of the sap A homologs is altered so that the 5' section of the homolog-specific region is replaced by a DNA cassette encoing a heterologous antigen.
  • the cassettes that can be inserted are immunogens related to Salmonella, Campylobacter jejuni, E. c ⁇ li 0157:H7, human immunoeficiency virus (HIV), simian immunoeficiency virus (SIV) as well as veneral, gastrointestinal, or respiratory pathogens of humans, cattle sheep, poultry, horses, swine, and reptiles .
  • the replacement is constructed so that the encoded S-layer protein is a bipartite chimera consisting a central region composed of the heterologous antigen and a C-terminal segment containing the S-layer protein secretion signal.
  • This protein would be able to be secreted from the cell by means of the C-terminal secretion signal thereby exposing the heterologous antigen to the host immune system.
  • the natural cassettes are replaced by the chimerae, one embodiment of the invention is the expectation that carriage of this organism is substantially shorter than for the wild type strain and thus, self-limiting. This strain should colonize for less than 3 months allowing the host to develop a mucosal and systemic immune response to each of the immunogens.
  • one may use a mutant . fetus strain in which all but one of the cassettes is replaced by a heterologous antigen.
  • This embodiment is similar to Example 12 except that one of the cassettes (e.g., sapA2 ) remains in its native configuration but that the others are mutagenized to form chimerae.
  • the advantage of this construction is that one can induce immunity to C. fetus based on the single full-length S- Layer protein produced.

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Abstract

Cette invention concerne une souche mutante de C. fetus dans laquelle chacune des cassettes est remplacée par un antigène hétérologue, une souche mutante de C. fetus dans laquelle toutes les cassettes sauf une sont remplacées par un antigène hétérologue, une souche mutante de C. fetus dans laquelle recA est mutagénisé et une souche de C. fetus dans laquelle recA est mutagénisé et l'homologue sapA exprimé est une chimère comprenant un peptide hétérologue.
PCT/US1998/001780 1997-01-31 1998-01-30 Procedes d'apport d'antigenes permettant d'effectuer des vaccinations avec un vecteur vivant WO1998033386A1 (fr)

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WO2004084936A3 (fr) * 2003-02-06 2005-06-16 Cerus Corp Microbes modifies vivant en milieu naturel, compositions de vaccins, et procedes d'utilisation correspondants
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003055906A1 (fr) * 2001-12-28 2003-07-10 Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno Proteines a couche de surface bacterienne modifiees
WO2004084936A3 (fr) * 2003-02-06 2005-06-16 Cerus Corp Microbes modifies vivant en milieu naturel, compositions de vaccins, et procedes d'utilisation correspondants
US7691393B2 (en) 2003-02-06 2010-04-06 Anza Therapeutics, Inc. Listeria attenuated for entry into non-phagocytic cells, vaccines comprising the Listeria, and methods of use thereof
US7695725B2 (en) 2003-02-06 2010-04-13 Aduro Biotech Modified free-living microbes, vaccine compositions and methods of use thereof
AU2004224425B2 (en) * 2003-02-06 2010-06-24 Aduro Biotech Modified free-living microbes, vaccine compositions and methods of use thereof
US7833775B2 (en) 2003-02-06 2010-11-16 Aduro Biotech Modified free-living microbes, vaccine compositions and methods of use thereof
US7927606B2 (en) 2003-02-06 2011-04-19 Aduro Biotech Modified free-living microbes, vaccine compositions and methods of use thereof
WO2005009463A3 (fr) * 2003-07-24 2005-06-02 Cerus Corp Vaccins contre les cellules presentatrices de l'antigene et methodes d'utilisation des vaccins
US7842289B2 (en) 2003-12-24 2010-11-30 Aduro Biotech Recombinant nucleic acid molecules, expression cassettes, and bacteria, and methods of use thereof

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