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S Chatfield

Publications and source records attributed to S Chatfield.

At least 37 records · Page 2Linked to original sources
PubMed1995

Expression of LacZ from the htrA, nirB and groE promoters in a Salmonella vaccine strain: influence of growth in mammalian cells.

Attenuated Salmonella strains are currently being evaluated as live vectors for the delivery of heterologous antigens to the mammalian mucosal and systemic immune systems. An approach to improving the stability of heterologous antigen expression during vaccination is to drive expression of the foreign protein from promoters, e.g. nirB, that become activated when Salmonella enter the host. Salmonella strains were constructed that harboured similar multicopy plasmids encoding the lacZ gene. In each strain, lacZ expression was driven from either the nirB, htrA or groE promoters. Expression of LacZ increased in all vaccine strains as they were shifted from conditions of low to high temperature. In addition, expression of lacZ driven from the htrA and nirB promoters significantly increased when the Salmonella entered eukaryotic cells, including macrophages. Expression of lacZ from the groE promoter was significantly elevated in macrophages but not in cells derived from epithelia. These promoters may be useful for optimising heterologous antigen expression within immune cells of the host.

Antigens↗
PubMed1995

Mucosal and systemic immunogenicity of a recombinant, non-ADP-ribosylating pertussis toxin: effects of formaldehyde treatment.

The effect of formaldehyde treatment on the mucosal and systemic immunogenicity of the genetically detoxified pertussis toxin (PT-9K/129G) was investigated. Groups of BALB/c were immunized intranasally (i.n.) or subcutaneously (s.c.) with untreated, lightly formaldehyde treated (LFT) or heavily formaldehyde treated (HFT) recombinant pertussis toxin (PT) mutant, PT-9K/129G. Intranasal immunization with native PT-9K/ 129G induced significant levels of anti-toxin antibodies in serum and IgA anti-toxin responses in nasal and lung lavages of these mice. Similar local and systemic responses were observed following intransal immunization with LFT toxin. However, i.n. immunization with HFT toxin failed to induce a local IgA response and elicited a much diminished anti-toxin response in the serum. In contrast, the total antibody response following s.c. immunization was not significantly affected. In addition, i.n. immunization with native PT-9K/129G induced low but detectable levels of toxin neutralizing antibodies in the serum. These results show that native PT-9K/129G protein acts as a mucosal immunogen in mice and that this activity is greatly diminished by HFT of the protein.

Administration, Intranasal↗
PubMed1995

Differential induction of carrier antigen-specific immunity by Salmonella typhimurium live-vaccine strains after single mucosal or intravenous immunization of BALB/c mice.

In this study, we constructed strain KR21 (chi 4550 delta cya delta crp delta asd/pYA292asd(+)-toxC+) and compared it with BRD847 (aroA aroD/pnirB-toxC) for the ability to induce humoral and cellular immunity after a single oral or intravenous immunization in 3- to 4-week-old BALB/c mice. ToxC-specific serum immunoglobulin G (IgG) was detectable in animals orally immunized with either BRD847 or KR21. However, after intravenous immunization, IgG was detected only in BRD847-immunized animals. Measurement of immunoglobin types IgG1 and IgG2a suggests that a Th1 cellular response is prominent after immunizations with either system. ToxC-specific IgA was detected in fecal and vaginal samples of animals immunized orally and intravenously with BRD847, while those immunized with KR21 failed to show fecal or vaginal IgA responses. Delayed-type hypersensitivity was used as a measure of induction of T-cell responses in vivo. Mice immunized either orally or intravenously with BRD847 showed significant ear swelling responses after ToxC injections, while KR21-immunized animals failed to show a cellular response. These data indicate that the aroA aroD/pnirB system holds greater potential for inducing global immunity after a single dose when directly compared with the balanced lethal system (delta cya delta crp delta asd/pYA292asd+).

Administration, Oral↗
PubMed1995

Biological and genetic characterization of TnphoA mutants of Salmonella typhimurium TML in the context of gastroenteritis.

TnphoA transposon insertion mutants of phoN-negative derivatives of Salmonella typhimurium TML (of human gastroenteritic origin) were selected by growing mutagenized recipient bacteria under a variety of growth conditions. Ninety-seven individual mutants, which expressed alkaline phosphatase, were collected and tested for their ability to invade HEp-2 cells. Seven smooth mutants had a reduced ability to invade HEp-2 cells, and three smooth mutants were consistently more invasive than their corresponding parental strains. One rough mutant was of similar invasiveness and two were of reduced invasiveness when compared with that of parental strains. The seven smooth hypoinvasive mutants, the three smooth hyperinvasive mutants, and the three rough mutant strains were tested for their abilities to invade ileal enterocytes by the rabbit ileal invasion assay described previously (3). All smooth mutants exhibited parental levels of invasiveness. The rough mutants were hypoinvasive in the rabbit ileal invasion assay. The HEp-2 system is therefore not a good predictor of behavior in gut tissue in this model. DNA sequences flanking the transposon were determined for five mutants which were hypoinvasive in the HEp-2 cell assay. The mutations were found to be insertions in two previously identified invasion genes, invG and invH, and in a gene not normally associated with invasion, pagC. These observations lead one to be cautious in the interpretation of the biological significance of data obtained from invasion of tissue culture monolayers when extrapolated to gut tissue.

Alkaline Phosphatase↗
PubMed1995

A mutant pertussis toxin molecule that lacks ADP-ribosyltransferase activity, PT-9K/129G, is an effective mucosal adjuvant for intranasally delivered proteins.

We examined the capacity of a genetically detoxified derivative of pertussis toxin (PTX), PT-9K/129G, to act as a mucosal adjuvant for an intranasally (i.n.) administered tetanus vaccine. Groups of mice were immunized i.n. with the nontoxic C-terminal 50-kDa portion of tetanus toxin (fragment C [Frg C]) either alone or mixed with PT-9K/129G, PTX, or cholera toxin (CT) or were immunized subcutaneously (s.c.) with an equivalent amount of Frg C adsorbed to alhydrogel. In response to a single immunization, mice receiving Frg C plus PT-9K/129G or CT i.n. and parenterally immunized mice developed high-titer (> 20,000) anti-Frg C antibodies, whereas mice immunized i.n. with Frg C plus PTX or with Frg C alone seroconverted only after being boosted. The serum anti-Frg C response was dominated by immunoglobulin G1 (IgG1) in mice immunized with Frg C plus PT-9K/129G, with Frg C plus PTX, or s.c. In contrast, IgG1, IgG2a, and IgG2b contributed almost equally to the Frg C response when CT was the adjuvant. Anti-Frg C IgE was detected only in the sera of mice immunized i.n. with Frg C plus PTX and immunized s.c. with Frg C plus alhydrogel. High levels of IgA antibodies were present in nasal lavage fluid from mice immunized i.n. with Frg C plus PT-9K/129G, PTX, or CT but not in that from mice given Frg C alone i.n. or parenterally. The mucosal adjuvanticity of PT-9K/129G was manifested in inbred as well as outbred mice. A single i.n. dose of Frg C plus either PT-9K/129G or PTX (with high specific activity) was sufficient to protect all immunized mice from tetanus toxin challenge, in contrast to the case for mice that received Frg C alone i.n. We conclude that the pertussis toxin analog PT-9K/129G, which is devoid of ADP-ribosyltransferase activity, is a potent mucosal adjuvant for vaccines delivered via the respiratory tract.

Adjuvants, Immunologic↗
PubMed1994

Construction, expression, and immunogenicity of multiple tandem copies of the Schistosoma mansoni peptide 115-131 of the P28 glutathione S-transferase expressed as C-terminal fusions to tetanus toxin fragment C in a live aro-attenuated vaccine strain of Salmonella.

Genetic fusions have been constructed between the highly immunogenic but atoxic fragment C of tetanus toxin and a guest peptide, aa115-131, from the protective 28-kDa glutathione S-transferase Ag of Schistosoma mansoni. Fusions have been assembled to express one, two, four, and eight tandem copies of the peptide. The recombinant vectors have been electroporated into the nonvirulent aroA strain of Salmonella typhimurium SL3261. The fusion proteins are soluble and stably expressed in Salmonella as evaluated by Western blotting with fragment C and glutathione S-transferase antisera. Mice have been immunized i.v. with a single dose of the live recombinant salmonellae. The strains are stable in mice and elicit Ab responses directed against fragment C, as determined by enzyme-linked immunosorbent assays. Ab responses were also detected against the guest peptide. The Ab responses improved dramatically toward the aa115-131 peptide with increasing copy number, with the octameric "repitope" fusion displaying the greatest potency. This approach may represent a general strategy for eliciting immune responses against peptides in live bacterial vaccines.

Amino Acid Sequence↗
PubMed1994

Construction, expression, and immunogenicity of the Schistosoma mansoni P28 glutathione S-transferase as a genetic fusion to tetanus toxin fragment C in a live Aro attenuated vaccine strain of Salmonella.

A vector has been constructed to allow genetic fusions of guest antigens via a hinge domain to the C terminus of the highly immunogenic C fragment of tetanus toxin. A fusion has been constructed with the gene encoding the protective 28-kDa glutathione S-transferase (EC 2.5.1.18) from Schistosoma mansoni. The recombinant vector has been electroporated into the nonvirulent Salmonella typhimurium aroA live vaccine strain SL3261. The corresponding chimeric protein is stably expressed in a soluble form in Salmonella as evaluated by Western blotting with fragment C and glutathione S-transferase antisera. Mice immunized intravenously with a single dose of the live recombinant bacteria elicit antibodies to both fragment C and glutathione S-transferase as detected by enzyme-linked immunosorbent assays. Furthermore, all of the mice were solidly protected when challenged with lethal doses of either tetanus toxin or the virulent Salmonella typhimurium strain C5. Mice have also elicited antibodies to fragment C and glutathione S-transferase after oral immunization. It may be that a live trivalent vaccine against typhoid, tetanus, and schistosomiasis is feasible.

Animals↗
PubMed1994

Molecular basis for Lewis alpha(1,3/1,4)-fucosyltransferase gene deficiency (FUT3) found in Lewis-negative Indonesian pedigrees.

The Le(a) and Le(b) human blood group antigens are synthesized in tissues producing exocrine secretions; they also circulate in plasma, where they are adsorbed by erythrocytes. They are synthesized by two fucosyltransferases, encoded by Lewis (FUT3) and secretor (FUT2) loci. This genetic model has been challenged because some erythrocyte Lewis-negative individuals express Lewis antigens in saliva. To define the molecular basis of this apparent discrepancy, we sequenced FUT3 in Lewis-negative individuals. We identified two single base pair changes. One, termed L1, yields a Leu-20-->Arg substitution in the enzyme's transmembrane domain. When expressed in COS-7 cells, enzyme substrate affinities are essentially identical to those of wild type. However, the mutant enzyme is found at substantially reduced levels in transfected cells. This suggests that the L1 mutation may alter the Golgi membrane anchoring of the enzyme. It was found alone in double dose in 10 of 30 erythrocyte Lewis-negative individuals, nine of whom express Lewis antigens in saliva. Therefore, L1 can account for erythrocyte/saliva-discrepant Lewis typing results. The L2 mutation creates an Ile-356-->Lys change in the enzyme's catalytic domain and inactivates the enzyme. It was found in double dose in 18 of 19 individuals bearing the double erythrocyte and salivary Lewis deficiency and can account for this phenotype.

Alleles↗
PubMed1994

Characterization of defined ompR mutants of Salmonella typhi: ompR is involved in the regulation of Vi polysaccharide expression.

The ompB operon, comprising the ompR and envZ genes, was cloned from a Salmonella typhi Ty2 cosmid bank and characterized by DNA sequence analysis. The S. typhi ompR and envZ genes contained open reading frames encoding proteins of 240 and 451 amino acids, respectively. Comparison with the Salmonella typhimurium OmpB protein sequences revealed 99.5% homology. The DNA sequence data were used to identify appropriate restriction sites for generating a defined deletion of 517 bp within the open reading frame of the ompR gene. This deletion was introduced by homologous recombination into the chromosomes of two S. typhi strains which already harbored defined deletions in both the aroC and aroD genes. The presence of the deletions within ompR was confirmed by Southern hybridization and sequencing of the DNA fragments surrounding the deleted regions by PCR. The S. typhi ompR mutants displayed a marked decrease in OmpC and OmpF porin expression as demonstrated by examination of outer membrane preparations. It was also found that S. typhi strains harboring the defined ompR deletions no longer agglutinated with Vi antiserum. However, when a functional ompB operon was introduced back into the S. typhi ompR mutants, either on a multicopy plasmid or as a single-copy chromosomal replacement, the Vi+ phenotype was restored. The levels of Vi synthesis were also found to be sensitive to different concentrations of sodium chloride present in the growth medium, although the levels of sensitivity varied between different isolates of S. typhi. It is therefore concluded that the ompR-envZ two component regulatory system plays an important role in the regulation of Vi polysaccharide synthesis in S. typhi and that one of the environmental signals for this regulation may be osmolarity.

Agglutination↗
PubMed1994

Successful transfusion in the presence of anti-K4 (anti-Kpb).

A 71-year-old group A, DC female, with anti-K4(-Kpb), -E, and -S was admitted for her second coronary artery surgery. Four units of autologous red blood cells (1BCs) were transfused perioperatively, and four units of homologous K:-4, E-, S- RBCs were transfused over the next 24 hours. Ten units of fresh frozen plasma and 28 units of platelets were also transfused. Continued bleeding necessitated calling donors from other states in AUStralia and from the International Panel of Donors of Rare Type, Bristol, UK, maintained by the World Health Organization. Four units of group 0, K:4, E-, S- KHCs were transfused in the next 24 hours while the K:-4 blood was in transit. No immediate signs of a transfusion reaction were noted. Blood pressure, temperature, bilirubin, and urinary output were normal, and an increase in hemoglobin was observed. A positive direct antiglobulin test was evident 3 days posttransfusion of the incompatible units and was still present 8 days later. Anti-A and anti-K4 were eluted from the patient's RBCs. The titer of anti-K4 increased from 8 preoperatively to 2,048 19 days posttransfusion of K:4 RBCs. Subsequent transfusions were not required, and the patient was discharged 3 weeks after the operation without further incident.

Journal Article↗
PubMed1994

The use of live attenuated Salmonella for oral vaccination.

Studies of the pathogenesis of Salmonella at the molecular level have led to the identification of several classes of genes that are involved in survival in the host. This has led to the availability of a panel of attenuating lesions which are now being used to develop several rationally attenuated strains which are being evaluated as oral vaccines against human and animal salmonellosis. Much effort has been directed towards the development of a more efficacious single dose oral typhoid vaccine and there are now several candidates in Phase 1 studies. The successful development of a genetically defined oral typhoid vaccine will not only be a major step forward in the control of typhoid but will pave the way for development of practical human vaccines based on using the strain to deliver heterologous antigens to the human immune system. We have concentrated on developing a single dose oral tetanus vaccine based on constructing strains expressing fragment C (a non-toxic immunogenic protein derived from tetanus toxin). Several different promotors have been used for controlling the expression of fragment C and these have been introduced into double aro mutants of S. typhimurium and compared for their ability to elicit protective immune responses in mice. This work has demonstrated that it is possible to protect mice against tetanus toxin challenge after a single oral dose of one of these recombinant Salmonella strains. Analogous hybrid S. typhi double aro mutants have now been constructed for potential use in humans.

Administration, Oral↗
PubMed1993

PERINAL--a new no-touch spray to relieve the symptoms of pruritus ani.

A new no-touch aqueous spray has been developed for the topical treatment of pruritus ani. It contains 0.2% hydrocortisone and 1.0% lignocaine hydrochloride, and has been shown in studies involving 83 patients to constitute a most convenient and clinically effective preparation for alleviating the symptoms of this distressing chronic condition.

Aerosols↗
PubMed1993

Protection of mice against respiratory Bordetella pertussis infection by intranasal immunization with P.69 and FHA.

Intranasal immunization of adult female Balb/c mice with the Bordetella pertussis antigens FHA or P.69, greatly enhanced their ability to clear B. pertussis from their lungs following aerosol challenge compared with ovalbumin-immunized controls. Low numbers of lymphocytes secreting antibodies (IgG, IgA and IgM) against the immunizing antigens could be isolated from the lungs of immunized mice. Following aerosol challenge with B. pertussis there was a large increase in the numbers of FHA or P.69-specific antibody-secreting cells in the lungs of mice immunized with these antigens. Intranasal immunization, particularly with FHA, also primed mice to develop a systemic serum anti-pertussis antibody response subsequent to challenge. However, pulmonary clearance of B. pertussis correlated most closely with the local antibody response. A strong anti-FHA response was demonstrated in the lungs of mice that received a booster dose of FHA 9 months after their previous exposure to FHA, demonstrating that long immunological memory can develop in the murine respiratory tract following direct application of pertussis antigens to the respiratory tract mucosa.

Adhesins, Bacterial↗
PubMed1993

The development of oral vaccines based on live attenuated Salmonella strains.

Safe, live attenuated Salmonella strains can be produced by introducing defined non-reverting mutations into the chromosome. Such rationally attenuated strains have proved to be excellent oral vaccines in several animal species and can therefore be considered as candidate vaccines against invasive salmonellosis in both animals and man. A panel of attenuating lesions is now available from which it is possible to tailor the level of attenuation and hence produce strains with different immunogenic properties. Because of the spectrum of immune responses produced by such Salmonella vaccine strains they have been utilised extensively as vectors for delivering heterologous antigens to the mammalian immune system. We have focussed on the development of a single dose oral tetanus vaccine based on attenuated Salmonella strains expressing a non-toxic, immunogenic protein derived from tetanus toxin (fragment C). Several different expression systems have been used for fragment C and candidate vaccine strains have been constructed that are capable of protecting orally immunised mice against a lethal challenge with tetanus toxin. An oral tetanus vaccine may help to reduce the mortality rate from tetanus in the developing world by overcoming the problems associated with the implementation of vaccine programmes using the current parenteral vaccine.

Administration, Oral↗
PubMed1992

Characterization of a Salmonella typhimurium aro vaccine strain expressing the P.69 antigen of Bordetella pertussis.

The P.69 Bordetella pertussis protective antigen was expressed by use of the trc promoter from the chromosome of a Salmonella typhimurium aro vaccine strain, BRD509, by integrating the prn gene, encoding the 93-kDa precursor of this protein, into the aroC locus. P.69 was detected on the cell surface of the S. typhimurium strain (BRD640) by agglutination and immunoelectron microscopy. BALB/c mice immunized orally or intravenously with BRD640 showed a significant level of protection against an aerosol challenge with virulent B. pertussis, compared with control animals. No anti-P.69 antibodies in the serum or anti-P.69 antibody-secreting cells in the lungs were detected in BRD640-vaccinated animals, although cells isolated from spleens showed a P.69-dependent cell proliferative response. In contrast, low levels of anti-P.69 antibodies in the serum and anti-P.69 antibody-secreting cells in the lungs were detected in immunized mice following a B. pertussis challenge.

Adhesins, Bacterial↗
PubMed1991

Construction of genetically defined double aro mutants of Salmonella typhi.

The construction of genetically defined, double aro mutant strains CVD906 and CVD908, which were derived from Salmonella typhi strain ISP1820 (a recent isolate of S. typhi from Chile) and from laboratory strain Ty2, respectively, is described. Strains CVD906 and CVD908 differ from previously described aro mutants of S. typhi as their aro deletion mutations do not extend beyond the limits of the mutated aro genes, and no antibiotic-resistance genes, plasmid sequences or S. typhimurium DNA sequences remain in the mutant strains. In minimal medium the aro mutants of S. typhi are unable to replicate whereas the wild type parent strains grow well in minimal medium. Using intraperitoneal inoculation of mice with S. typhi strains suspended in hog gastric mucin as a virulence assay, it is shown that the single aro mutants and the double aro mutants of Ty2 and ISP1820 are attenuated in mice. Trans complementation of the aro mutants with the aroC gene or aroD gene, or both, results in strains that are phenotypically identical to that of the wild type parents indicating that no measurable additional changes other than loss of the aro gene function occurred during strain construction.

Adolescent↗
PubMed1991

Molecular cloning and characterization of the aroD gene encoding 3-dehydroquinase from Salmonella typhi.

The aroD gene from Salmonella typhi, encoding 5-dehydroquinate hydrolyase (3-dehydroquinase), has been cloned into Escherichia coli and the DNA sequence determined. The aroD gene was isolated from a cosmid gene bank by complementation of an S. typhimurium aroD mutant. Analysis of the DNA sequence revealed the presence of an open reading frame capable of encoding a protein of 252 amino acids with a calculated Mr of 27706. Comparison of the deduced S. typhi 3-dehydroquinase protein sequence with that elucidated for E. coli revealed 69% homology. Alignment of the S. typhi sequence and equivalent Aspergillus nidulans and Saccharomyces cerevisiae sequences showed that homology was lower, at 24%, but still significant. Use of a minicell expression system demonstrated that a polyclonal antibody raised against E. coli 3-dehydroquinase cross-related with its S. typhi counterpart.

Amino Acid Sequence↗