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Biomedical subjects

G Dougan

Publications and source records attributed to G Dougan.

At least 217 records · Page 12Linked to original sources
PubMed1990

Anti-viral immunity induced by recombinant nucleoprotein of influenza A virus. III. Delivery of recombinant nucleoprotein to the immune system using attenuated Salmonella typhimurium as a live carrier.

A plasmid encoding the influenza nucleoprotein gene from A/NT/60/68 virus was transduced into the attenuated Salmonella typhimurium aroA- strain SL3261. The bacterial vector expressing the viral gene product was able to induce both humoral and cell-mediated immune responses to the nucleoprotein antigen. CD4+ virus-specific T cells capable of proliferation were readily induced and, in some circumstances, class II major histocompatibility complex (MHC)-restricted cytotoxicity was detected. However, virus-specific class I MHC-restricted cytotoxic T lymphocytes (CTL) were not detected after such immunization. Mice immunized orally with the nucleoprotein-expressing bacteria mounted a strong anti-viral antibody response and spleen cells from such mice proliferated specifically to virus challenge in vitro, producing interferon-gamma (IFN-gamma) and interleukin-2 (IL-2). Orally immunized mice showed significant protection from challenge infection with influenza virus if the mice were also boosted intranasally before infection.

Animals↗
PubMed1990

Anti-viral immunity induced by recombinant nucleoprotein of influenza A virus. II. Protection from influenza infection and mechanism of protection.

Protection against influenza A virus infection in mice immunized with recombinant nucleoprotein (rNP) was studied. Nucleoprotein-immune mice were protected against a lethal challenge with A/Puerto Rico/8/34 (A/PR8) virus but showed considerable morbidity before recovery. Local boosting of the immune system with rNP by intranasal immunization improved the protection in NP-immune mice, and the decrease in morbidity after infection was reflected in accelerated clearance of virus from lungs. However, immune, boosted mice also rapidly cleared an antigenically unrelated influenza B virus from their lungs. Mice immunized with rNP precipitated with alhydrogel, that subsequently developed significant resistance to virus infection, failed to generate detectable levels of class I major histocompatibility complex (MHC)-restricted killer cells. Furthermore, B10.A(5R) mice that are non-responders for NP-specific class I killer cells could also be protected by immunization with rNP. In contrast, rNP-immunized mice developed strong proliferative T-cell responses to rNP. These data argue for an important role for helper T cells rather than virus-specific class I cytotoxic T cells in protection against influenza virus infection induced by rNP.

Animals↗
PubMed1989

Cloning and characterisation of the serC and aroA genes of Yersinia enterocolitica, and construction of an aroA mutant.

A gene library of Yersinia enterocolitica 8081 was constructed in the cosmid vector pHC79. Recombinants containing the aroA gene, encoding 5-enolpyruvylshikimate 3-phosphate synthase, were identified by complementation of the aroA mutation in Escherichia coli K-12 strain AB2829. All six recombinant plasmids which complemented aroA also complemented the serC mutation in E. coli K-12 strain KL282. Tn5 mutagenesis suggested serC encoding 3-phosphoserine aminotransferase was the proximal gene in an operon with aroA. The nucleotide sequence of a 3-kb HindII-EcoRV fragment encoding the two genes was determined. The serC and aroA open reading frames contain 362 and 428 codons, respectively, and the deduced amino acid sequences share 78% and 81% homology, respectively, with the corresponding E. coli genes. Sequence inspection revealed no obvious terminators or promoters in the intergenic region. The cloned Y. enterocolitica aroA gene was inactivated in vitro and reintroduced into the parental Y. enterocolitica 8081 strain using the suicide vector pJM703.1. Stable aroA insertion mutants of Y. enterocolitica were isolated.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1989

Bacteriophage P22 as a vehicle for transducing cosmid gene banks between smooth strains of Salmonella typhimurium: use in identifying a role for aroD in attenuating virulent Salmonella strains.

A cosmid gene bank of the virulent Salmonella typhimurium C5 was constructed in Escherichia coli K12. The bank was repackaged into bacteriophage heads and transduced into the semi-rough S. typhimurium strain AS68 which expresses the LamB lambda receptor protein. Approximately 6000 ampicillin-resistant transductants were pooled and used as host for the propagation of bacteriophage P22. The P22 lysate was able to transduce cosmid recombinants to smooth strains of S. typhimurium and individual transductants were selected which complemented various S. typhimurium auxotrophic mutations. A stable mutation was introduced into the aroD gene of S. typhimurium C5. The resulting aroD- mutant, named CU038, was highly attenuated compared with the wild-type parent strain and BALB/c mice immunised orally with CU038 were well protected against challenge with the virulent C5 parental strain. Using the cosmid bank repackaged into bacteriophage P22 heads it was possible to isolate cosmid recombinants that could complement the aroD mutation of CU038 either by in vitro selection using minimal medium or in vivo selection for restoration of virulence in BALB/c mice. Repackaged P22 cosmid banks could provide a simple system for selecting in vivo for Salmonella virulence determinants. A Salmonella typhi strain harbouring mutations in aroA and aroD was constructed for potential use as a live oral typhoid vaccine in humans.

Animals↗
PubMed1989

Live Salmonella as vaccines and carriers of foreign antigenic determinants.

Salmonella species can be rationally attenuated by introducing non-reverting defined mutations into the genome to produce live vaccine strains. Several genes have been identified which when mutated, will attenuate Salmonellae. In particular, salmonella strains harbouring mutations in genes involved in the pre-chorismate biosynthetic pathway make excellent oral vaccines evoking strong humoral, local and cellular immune responses in the host. Because of the spectrum of immune responses induced by live vaccine strains they have the potential to be used for delivery of heterologous antigens to the mammalian immune system. A number of antigens from other bacteria, viruses and parasites have been expressed in live salmonella vaccine strains. Such hybrid strains have the potential to be used as multivalent vaccines against a number of infectious diseases.

Bacterial Vaccines↗
PubMed1989

Molecular cloning and characterization of protective outer membrane protein P.69 from Bordetella pertussis.

Protein P.69 is localized on the outer membrane of Bordetella pertussis and is one of the virulence factors believed to contribute to the disease state of whooping cough. We demonstrate that protein synthesis of P.69 is under genetic control of the vir locus. Using oligonucleotide probes derived from the protein sequence of a cyanogen bromide fragment, we have cloned the gene for P.69 from B. pertussis CN2992. Analysis of the DNA sequence reveals a G + C-rich gene capable of encoding a protein of 910 amino acids with a Mr of 93,478, suggesting that P.69 is a processed form of a larger precursor. In common with some of the genes in the pertussis toxin operon, the sequence CCTGG was found 5' to the ATG initiation codon. At the 3' end, 29 bases after the TAA stop codon, the sequence GTTTTTCCT was found and may have some function in transcription termination. A full-length clone of the gene for P.69 carried by the cosmid pBPI69 was unable to direct the expression of P.69 protein in an Escherichia coli host. The generation of P.69-fusion products allowed the detection of P.69-specific protein products synthesized in E. coli.

Amino Acid Sequence↗
PubMed1989

Phospholipase C and haemolytic activities of Clostridium perfringens alpha-toxin cloned in Escherichia coli: sequence and homology with a Bacillus cereus phospholipase C.

The Clostridium perfringens alpha-toxin (phospholipase C) gene (cpa) has been cloned and expressed in Escherichia coli. The biological activities of the cloned gene product have been analysed and the complete nucleotide sequence of the cpa gene has been determined. The cloned cpa gene product, which is exported to the periplasm in E. coli, possesses both phospholipase C and haemolytic activities. Haemolysis is not apparent when cell extracts are incubated with isotonic suspensions of sheep erythrocytes, but can be detected and quantified readily when dilutions of the same extracts are placed in wells in sheep-blood agar plates. Like other sequenced clostridial genes, the cpa gene has a high AT content (66.4%), exhibits a strong bias for using codons with A or T in the wobble position, and the 350 base pairs upstream from the gene have a significantly higher AT content (79.5%) than the coding region. The cpa gene encodes a 398 amino acid polypeptide with a deduced molecular weight of 45,481 D. This is very similar to the estimated molecular weight (Mr) of the cpa primary gene product expressed in an in vitro transcription-translation system (Mr 46,000), but larger than the cpa gene product detected in E. coli minicells, E. coli whole cells or in C. perfringens cells (Mr 43,000), suggesting post-translational processing. The 28 N-terminal residues of the deduced alpha-toxin sequence possess the consensus features of a signal peptide and may be removed during secretion. The deduced alpha-toxin sequence shares significant structural homology with the phosphatidylcholine-preferring phospholipase C of Bacillus cereus.

Amino Acid Sequence↗
PubMed1989

Virulence, persistence, and immunogenicity of Yersinia enterocolitica O:8 aroA mutants.

The virulent Yersinia enterocolitica strain 8081 killed BALB/c mice within 5 days of oral infection with a 50% lethal dose of log10 7.1, whereas an aroA mutant of 8081, YAM.1, and the plasmidless variant 8081c failed to kill mice. Unlike 8081, YAM.1 and 8081c did not persist or grow in the Peyer's patches, mesenteric lymph nodes, livers, or spleens of mice. Mice immunized orally with single doses of live YAM.1 were poorly protected against a lethal 8081 challenge, whereas mice immunized with three doses of YAM.1 were moderately well protected.

Administration, Oral↗
PubMed1989

Characterization of porin and ompR mutants of a virulent strain of Salmonella typhimurium: ompR mutants are attenuated in vivo.

The ompC, ompD, and ompF genes encode the three major porins of Salmonella typhimurium. ompR encodes a positive regulator required for the expression of ompC and ompF. Transposon-generated mutations in ompC, ompD, ompF, and ompR were introduced into the S. typhimurium mouse virulent strain SL1344 by P22-mediated transduction. Following preliminary characterization in vitro, the strains were used to challenge BALB/c mice by using the oral or intravenous route. Strains harboring ompC or ompF mutations were as virulent as SL1344 after oral challenge. Strains harboring ompD mutations had a slight reduction in virulence. In contrast, ompR mutants failed to kill BALB/c mice after oral challenge and the intravenous 50% lethal dose was reduced by approximately 10(5). The ompR mutants persisted in murine tissues for several weeks following oral or intravenous challenge. Furthermore, mice orally immunized with these ompR mutant strains were well protected against challenge with virulent SL1344.

Administration, Oral↗
PubMed1989

Isolation of orally attenuated Salmonella typhimurium following TnphoA mutagenesis.

One hundred fifty Tn5 IS50L::phoA (TnphoA) mutants of a mouse-virulent, nalidixic acid-resistant (Nalr), prototrophic Salmonella typhimurium strain, C5 Nalr, were isolated. None of the mutants were auxotrophs. Groups of 8 to 10 BALB/c mice were infected orally with each of 95 mutants with a dose equivalent to 20-fold the 50% lethal dose of the wild-type C5 Nalr strain, and deaths were counted over the next 28 days. Fifteen of the mutants failed to kill any mice, whereas all mice died following challenge with the other mutants. Nine of the 15 attenuated mutants exhibited a defect in lipopolysaccharide biosynthesis. The remaining six mutants were smooth. The TnphoA transposon of each of the smooth attenuated mutants was moved, using P22-mediated transduction, into a fresh C5 background, and all retransductants were still attenuated. Analysis of the membrane proteins of the attenuated mutants failed to reveal any alterations in detectable major outer membrane proteins, although colonies of two of the mutants exhibited a mucoid phenotype following growth on L-agar plates. Individual attenuated mutants differed in their abilities to translocate to livers and spleens of mice following oral infection. All of the smooth TnphoA mutants exhibited increased 50% lethal doses with respect to the wild type following intravenous infection of BALB/c mice. Southern analysis of DNA prepared from each of the mutants suggested that TnphoA had inserted into a number of different sites in the S. typhimurium genome. None of the TnphoA mutants had inserts in the virulence-associated plasmid.

Administration, Oral↗
PubMed1988

Antiviral immunity induced by recombinant nucleoprotein of influenza A virus. I. Characteristics and cross-reactivity of T cell responses.

The gene for the nucleoprotein of the A/NT/60/68 influenza virus was expressed in bacteria and the recombinant protein purified. Lymph node cells from mice immunized with recombinant nucleoprotein proliferated in response to in vitro stimulation with a range of type A influenza viruses. Proliferation was inhibited by mAb to CD4 and class II MHC gene products. IFN-gamma was produced and type-specific CTL were generated in stimulated cultures of immune lymph node cells. These CTL were CD4+ and restricted to class II MHC gene products. Immunization with recombinant nucleoprotein generated Th cells in vivo as measured by the ability to generate an accelerated response to hemagglutinin after challenge with inactivated virus. The results are discussed with reference to a cross-reactive vaccine against influenza.

Animals↗
PubMed1988

Oral vaccination.

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Administration, Oral↗
PubMed1988

Construction and characterization of vaccine strains of Salmonella harboring mutations in two different aro genes.

Derivatives of the mouse-virulent Salmonella typhimurium strain SL1344 were constructed harboring stable mutations in aroC alone or in aroC and aroA together. Fifty percent lethal doses after intravenous inoculation of the mutants into BALB/c mice were determined, and the mutants were as highly attenuated as were SL1344 aroA derivatives. All aro-dependent derivatives persisted in vivo at similar levels and for similar intervals in the livers and spleens of BALB/c mice infected intravenously. Mice vaccinated orally with 10(10) live organisms of the different derivatives survived and were well protected against oral challenge with virulent S. typhimurium. A Salmonella typhi Ty2 derivative, designated WBL2000, was constructed that harbors well-defined, stable mutations in both aroA and aroC.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1988

Identification and characterization of TnphoA mutants of Salmonella that are unable to pass through a polarized MDCK epithelial cell monolayer.

Surface protein mutants of the invasive Salmonella species, S. choleraesuis, were generated using the transposon TnphoA. 626 alkaline phosphatase (PhoA+) fusion mutants were identified and screened for their ability to pass through (transcytose) polarized epithelial monolayers of Madin Darby canine kidney (MDCK) cells grown on membrane filters. Forty two mutants were unable to pass through this barrier. All of these transcytosis mutants were unable to adhere to or invade MDCK monolayers, yet these mutations were not in the genes encoding type 1 pili or mannose-resistant haemagglutination (MRHA). These transcytosis mutants could be grouped into six classes. Class 1 mutants had altered lipopolysaccharide (LPS) O side-chain structures while Class 2 mutants had defects in their LPS core. Mutants belonging to Classes 5 and 6 did not decrease the transepithelial electrical resistance of polarized MDCK cell monolayers, in contrast to the parental strain and the other mutants (Classes 1, 2, 3 and 4). Mutants belonging to Class 1 were less virulent in mice, while Class 2 (defective core) and Classes 4 and 5 (normal LPS) mutant strains were avirulent in mice. Mutants from Classes 3 and 6 were as virulent in mice as S. choleraesuis. These results suggest that the ability to pass through epithelial barriers may be an important virulence characteristic of Salmonella. These data indicate that bacterial adherence, internalization and monolayer transcytosis are closely linked events. It was also demonstrated that a mutant with decreased rates of intracellular replication still passed through the monolayer at rates similar to wild-type S. choleraesuis.

Alkaline Phosphatase↗
PubMed1988

Characterization of aromatic- and purine-dependent Salmonella typhimurium: attention, persistence, and ability to induce protective immunity in BALB/c mice.

Stable transposon-generated auxotrophic mutations in aroA, purA, and purE or aroA and purA together were introduced into Salmonella typhimurium strains which were virulent in mice. Strains harboring any of these mutations were attenuated when tested in BALB/c mice. purE strains were less attenuated than aroA or purA strains. Both aroA and purA mutants persisted for several weeks in the livers and spleens of the mice after intravenous infection, although the numbers of viable cells detected at various times after infection differed. aroA strains persisted at a higher level than purA strains and were effective live vaccines given intravenously or orally. purA strains were ineffective as oral vaccines and were poor intravenous vaccines. Strains harboring both aroA and purA mutations together were ineffective vaccines when administered orally or intravenously even though they persisted in the livers and spleens of the mice for long periods after intravenous infection.

Administration, Oral↗