Steven Chatfield

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.

Evaluation of Salmonella typhimurium strains harbouring defined mutations in htrA and aroA in the murine salmonellosis model

Derivatives of the mouse-virulent Salmonella typhimurium strain SL1344 were constructed harbouring defined mutations in htrA, aroA or htrA aroA combined. When administered orally or intravenously to BALB/c mice, all the mutants were found to be highly attenuated. All mutants were able to confer significant protection against lethal challenge with SL1344 after a single oral dose of live organisms. SL1344 htrA mutants persisted in livers and spleens at a lower level than SL1344 aroA mutants after intravenous administration. SL1344 htrA aroA mutants persisted at an even lower level and were cleared from the livers and spleens of mice within 21 days of intravenous administration. Thus htrA and htrA aroA mutants can be considered as potential oral vaccines against salmonellosis.

Characterization of Salmonella enterica Derivatives Harboring Defined aroC and Salmonella Pathogenicity Island 2 Type III Secretion System (ssaV) Mutations by Immunization of Healthy Volunteers

ABSTRACT The attenuation and immunogenicity of two novel Salmonella vaccine strains, Salmonella enterica serovar Typhi (Ty2 ΔaroC ΔssaV, designated ZH9) and S. enterica serovar Typhimurium (TML ΔaroC ΔssaV, designated WT05), were evaluated after their oral administration to volunteers as single escalating doses of 107, 108, or 109 CFU. ZH9 was well tolerated, not detected in blood, nor persistently excreted in stool. Six of nine volunteers elicited anti-serovar Typhi lipopolysaccharide (LPS) immunoglobulin A (IgA) antibody-secreting cell (ASC) responses, with three of three vaccinees receiving 108 and two of three receiving 109 CFU which elicited high-titer LPS-specific serum IgG. WT05 was also well tolerated with no diarrhea, although the administration of 108 and 109 CFU resulted in shedding in stools for up to 23 days. Only volunteers immunized with 109 CFU of WT05 mounted detectable serovar Typhimurium LPS-specific ASC responses and serum antibody responses were variable. These data indicate that mutations in type III secretion systems may provide a route to the development of live vaccines in humans and highlight significant differences in the potential use of serovars Typhimurium and Typhi.

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 mono‐layers, 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.

Attenuated Salmonella as live oral vaccines against typhoid fever and as live vectors.

Attenuated Salmonella typhi vaccine strain CVD 908, which harbors deletion mutations in aroC and aroD, has been shown to be well-tolerated and highly immunogenic, eliciting impressive serum antibody, mucosal IgA and cell-mediated immune responses. A further derivative prepared by introducing a deletion in htrA (which encodes a heat-shock protein that also has activity as a serine protease in CVD 908 (Chatfield et al., unpublished data) resulted in CVD 908-htrA. In phase 1 clinical trials, CVD 908-htrA appears very attractive as a live oral vaccine candidate. Both CVD 908 and CVD 908-htrA are useful as live vector vaccines to deliver foreign antigens to the immune system. Conditions that enhance the expression and immunogenicity of foreign antigens carried by CVD 908 and CVD 908-htrA are being investigated.

Immunisation of mice using Salmonella typhimurium expressing human papillomavirus type 16 E7 epitopes inserted into hepatitis B virus core antigen

Live vaccines based on BRD509, an attenuated S. typhimurium (aroA, aroD) strain, were constructed that directed the expression of hepatitis B core antigen particles (HBcAg) (BRD969) or HBcAg harbouring human papillomavirus type 16 E7 protein sequences (BRD974), under the control of the in vivo inducible nirB promoter. These strains were used to orally or intravenously immunise different inbred mouse strains and humoral, secretory and cellular anti-E7 and anti-HBcAg responses were monitored. Both BRD969 and BRD974 induced anti-HBcAg humoral IgG responses following oral or intravenous immunisation of B10 mice, although responses were higher in BRD969 immunised animals. IgG subclass analysis revealed a predominantly IgG2a response in these animals. BRD974, but not BRD969, induced anti-E7 humoral IgG responses. Anti-HBcAg (BRD969 and BRD974) and anti-E7 (BRD974) IgA responses were detected in the intestines of orally immunised mice. Anti-Salmonella but not anti-HBcAg or anti-E7 T helper cell responses were detected in mice immunised with BRD509, BRD969 and BRD974. Thus Salmonella vaccine strains can be used to efficiently deliver HBcAg and E7 epitopes to the mucosal and systemic immune systems.

The Role of Th1 and Th2 Cells for Mucosal IgA Responsesa

We have used cytokine-knockout mice to help determine the precise requirements for CD4+ Th cell regulation of IgA responses. In these studies, we have used two different oral delivery systems to induce mucosal and systemic antibody responses to the vaccine TT. In normal mice, oral administration of TT with CT as adjuvant induces Th2 cells and cytokines, which give rise to mucosal IgA and serum IgG1, IgA, and IgE responses. On the other hand, oral immunization with rSalmonella expressing Tox C results in Th1-type responses as well as Th2 cell-derived IL-10 and macrophage-derived IL-6, which correlate with mucosal IgA and serum IgG2a antibody responses. Two major conclusions can be drawn from our studies with these two regimens in normal, IFN-gamma-/-, and IL-4-/- mice. First, oral administration of rSalmonella, which elicits classical Th1-type responses also induces significant mucosal IgA responses when given to mice with defective Th1- (IFN-gamma-/-) or Th2- (IL-4-/-) cytokine pathways. Interestingly, we detect Th2-type cells producing IL-10 and macrophage-secreting IL-6 in both normal and cytokine-deficient mice, and we postulate that these two cytokines are of most importance for murine IgA responses. Second, oral administration of TT plus CT as adjuvant induces classical Th2-type responses in both normal and IFN-gamma-/- mice. Further, lack of IL-4 results in failure to induce mucosal IgA responses. Thus, the IL-4 pathway is necessary for the CT adjuvant effect for mucosal IgA responses after oral immunization with a protein vaccine.

Role of the Bordetella pertussis P.69/pertactin protein and the P.69/pertactin RGD motif in the adherence to and invasion of mammalian cells

The role of the Bordetella pertussis P.69/pertactin protein in mammalian cell adhesion and invasion was investigated. Salmonella strains expressing surface-associated P.69/pertactin from a chromosomally located prn gene were significantly more invasive than isogenic parental strains. This effect was most pronounced in the poorly invasive, semi-rough S. typhimurium strain LB5010. Escherichia coli K-12 strain HB101 harbouring the plasmid p41869D, which encodes the full-length prn gene under the control of the tac promoter on the broad-host-range plasmid pMMB66EH, was significantly more adhesive to HEp-2 and Chinese Hamster Ovary (CHO) cells growing in culture than E. coli HB101(pMMB66EH). However, the ability of E. coli to invade mammalian cells was not affected by P.69/pertactin expression. P.69/pertactin-mediated adhesiveness of HB101 to HEp-2 and CHO cells was not influenced by the viability of the bacterial cells. However, adherence was markedly reduced when assays were performed for less than 3 h, at 4 degrees C or in the presence of cycloheximide, suggesting the active participation of the eukaryotic cell in bacterial adhesion. Site-directed mutagenesis was used to mutate Asp to Glu in an Arg-Gly-Asp (RGD-->RGE) sequence present in mature P.69/pertactin and the mutated gene was cloned in the same broad-host-range vector (plasmid p41869E). This mutation had no detectable influence on the ability of P.69/pertactin to mediate adhesion of HB101 to HEp-2 or CHO cells. Plasmids p41869D and p41869E were introduced into the bvg-negative B. pertussis strain BP347. Expression of P.69RGD or P.69RGE did not enhance the adhesiveness of BP347 for epithelial (HEp-2 and CHO) cells.

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.

The Novel Oral Typhoid Vaccine M01ZH09 Is Well Tolerated and Highly Immunogenic in 2 Vaccine Presentations

BACKGROUND M01ZH09 (Salmonella enterica serovar Typhi [Ty2 aroC(-) ssaV(-)] ZH9) is a live oral-dose typhoid vaccine candidate. M01ZH09 was rationally modified with 2 independently attenuating mutations, including a novel mutation in Salmonella pathogenicity island (SPI)-2. We demonstrate that M01ZH09, in a single oral dose, is well tolerated and prompts broad immune responses, regardless of whether prevaccination with a bicarbonate buffer is given. METHODS Thirty-two healthy adult subjects were randomized and given 5x109 cfu of M01ZH09, with (presentation 1) or without (presentation 2) prevaccination with a bicarbonate buffer. Immunogenicity data included Salmonella Typhi lipopolysaccharide (LPS)-specific immunoglobulin (Ig) A antibody-secreting cells (enzyme-linked immunospot [ELISPOT] assay), IgG serologic responses to Salmonella Typhi LPS, lymphocyte proliferation, and interferon (IFN)- gamma production. RESULTS The vaccine was well tolerated; adverse events after vaccination were mild. No fever or prolonged vaccine shedding occurred. Immunogenicity data demonstrated that 88% and 93% of subjects who received presentation 1 and presentation 2, respectively, had a positive response by ELISPOT assay; 81% of subjects in both groups underwent IgG seroconversion on day 14. Both groups had similar cellular immune responses to presentation 1 and presentation 2; lymphocyte proliferation to Salmonella Typhi flagellin occurred in 63% and 67% of subjects, respectively, and 69% and 73% of subjects, respectively, had an increase in IFN- gamma production. CONCLUSION The oral typhoid vaccine M01ZH09 is well tolerated and highly immunogenic in a single oral dose, with and without prevaccination with a bicarbonate buffer. Field studies to demonstrate protective efficacy are planned.