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The incidence of non-responders to hepatitis B (HB) virus SHBs antigen (Ag) vaccines has prompted the development of pre-S containing vaccines. The aim of this study was to characterise the murine immune response to a novel recombinant particle (Hepagene) (Medeva plc) containing pre-S1, pre-S2 and SHBsAg components. Hepagene induced potent in vitro spleen T-cell proliferative responses in both BALB/c (maximum stimulation index (SI) = 38) and SWR/J (maximum SI = 43) strains of mouse, following immunisation. High concentrations of interferon-gamma and low concentrations of interleukin-10 were detected in the media of spleen cells stimulated with Hepagene. The anti-Hepagene antibody response was higher in SWR/J mice and alhydrogel adjuvant significantly improved the titres. Anti-pre-S1 antibody was detected in both strains of mouse, whereas antipre-S2 antibody was only detected in SWR/J mice. IgG subclass analysis of the anti-Hepagene response revealed a Th2-type response in BALB/c mice and a mixed Th1/Th2 response in SWR/J mice. Hepagene induced higher anti-SHBs antibody responses than Engerix-B (11097 and 1276 IU/ml, respectively) in BALB/c mice. Hepagene therefore, stimulates strong cellular and humoral immune responses in murine models. The high anti-SHBs antibody response suggests that Hepagene is an improved hepatitis B virus vaccine.

Modifying bacterial virulence genes to probe the nature of host immunity is mostly unexplored. Here we investigate whether host immune responses can be regulated by modification of bacterial virulence genes. In mice, attenuated Salmonella mutant strains with clinical relevance elicited differential host immune responses. Oral administration of a mutant strain with a PhoP-null phenotype promoted potent innate immune responses of macrophages that were sufficient for host defense. In contrast, administration of an Aro- mutant strain elicited stronger specific antibody and T-helper (Th)-cell responses, wherein Th1-type cells were required for clearance. Thus, genetic manipulation of bacteria may be used to broadly alter immune mechanisms that regulate attenuation within the host and to tailor host immunity to specific bacterial pathogens.

Purpose/objectives: To determine if clots are present in the initial 10 ml of blood routinely discarded from venous access devices (VADs) prior to blood sampling, and to determine if clots form in the discard blood specimen during the five minutes required to complete blood specimen sampling., Design: A pretest/post-test design., Setting: A large, mid-Atlantic research institution., Sample: A convenience sample of 50 adult patients with cancer (27 males and 23 females) with a median age of 60. A large sampling size variation existed among the different VADs., Method: Two 5 ml discard specimens were drawn into separate syringes. Syringe #1 was filtered immediately, and syringe #2 was filtered after a five-minute dwell time. Both samples were filtered through a 40 micron filter., Main Research Variable: The presence or absence of clots., Findings: Fifty percent (n = 25) of the VADs had clots present on the filter from syringe #1. The clots varied in length, width, depth, and diameter, which precluded a consistent measurement. The investigators were able to measure either the diameter or length, depending on the shape of the clots. The majority of the clots (n = 17) appeared to be shaped like the lumen of a catheter and varied from 0.1 cm to 1.2 cm in length. Six clots were round and varied in diameter from 1.6 mm to 2.8 mm. Only 4% (n = 2) of the VADs had clots in syringe #2, but those clots were much larger, measuring 8.3 mm and 18.4 mm., Conclusions: The study addresses concerns of the investigators regarding the clinical practice of reinfusing discard blood obtained from VADs. Whether the clots present in the catheter and their reinfusion represent a significant risk to patient outcome is unclear., Implications for Nursing Practice: Until further research is conducted and the degree of risk can be better defined, methods of drawing blood that require reinfusion of discard blood from VADs are not recommended.

The genes encoding murine macrophage migration inhibitory factor (MIF), IL-2, IFN-gamma or TNF-alpha were cloned individually into an expression plasmid under the control of the inducible promoter nirB and transfected into the aroA- aroD- deletion mutant strain of Salmonella typhimurium (BRD509). These S. typhimurium derivatives (henceforward called constructs and termed GIDMIF, GIDIL2, GIDIFN and GIDTNF) expressed their respective cytokines in vitro under anaerobic conditions and stably colonized BALB/c mice up to 14 days after oral administration. The highly susceptible BALB/c mice that had received the constructs orally and that had been subsequently infected via the footpad with Leishmania major, developed significantly reduced disease compared with control mice administered the untransfected Salmonella strain (BRD509). Importantly, a combination of GIDMIF, GIDIFN, and GIDTNF administered orally after L. major infection was able to significantly limit lesion development and reduced parasite loads by up to three orders of magnitude. Spleen and lymph node cells of mice administered this combination expressed markedly higher levels of inducible nitric oxide synthase (iNOS) compared with those from mice receiving an equivalent dose of the control strain of Salmonella (BRD509). These data therefore demonstrate the feasibility of therapeutic treatment in an infectious disease model using cytokines delivered by attenuated Salmonella. The protective effect observed correlates with the induction of inducible nitric oxide synthase in vivo.

Salmonella aro vaccines are able to confer solid protection against homologous virulent challenge in several animal species. Calves were protected against virulent S. typhimurium challenge following administration of a single oral dose of live BRD562 vaccine. Immune responses elicited by the S. typhimurium aro vaccine strain BRD562 were studied following administration to calves by either the oral or subcutaneous route. Serum antibodies to Salmonella polypeptides, following oral or subcutaneous vaccination, were detected by immunoblotting and the route of inoculation found to affect both the antibody isotype and the antigens detected. Oral, but not subcutaneous, immunisation induced bovine serum IgA antibodies against Salmonella antigens of 30 kDa and 65 kDa and bovine IgG2 antibodies against a 35 kDa antigen. Subcutaneous vaccination triggered responses against antigens of 52 kDa, 54 kDa and 57 kDa which were not detected by immune plasma of animals immunised orally. Antibody responses to LPS were poor in animals inoculated by either route. Subcutaneous vaccination elicited T-cell responses against Salmonella antigens as measured by in vitro peripheral blood cell thymidine incorporation. These studies show that the S. typhimurium vaccine strain BRD562 is capable of inducing both humoral and cellular immune responses. Further studies are necessary to identify the nature of the antigens responsible for protection. Oral or subcutaneous inoculation of BRD562(pTETnir15) failed to induce serum antibodies against the fragment C of tetanus toxin (TetC) but was effective in mice. Oral vaccination with this recombinant vaccine induced mucosal IgA against TetC. This is the first time that Salmonella recombinant vaccines have been shown to successfully elicit antibodies against a guest antigen in cattle after one single oral inoculation.

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.

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.

We report the expression of fragment C of tetanus toxin (FC) fused to the eukaryotic cell binding domain (the carboxyl-terminus) of diphtheria toxin (FC-bDt fusion) in attenuated Salmonella typhi live vector vaccine strain CVD 908. The FC-bDt protein fusion was constructed using plasmid pTETnir15 which carries the gene encoding FC under control of the nirB promoter (nirBP). The open reading frame for FC was modified to incorporate an in-frame glycine-proline hinge region and a set of four restriction sites at the 3' end of the FC gene. A 482 bp DNA fragment encoding the eukaryotic cell binding domain of diphtheria toxin was then inserted at the 3' end of the modified FC gene to create an in-frame FC-bDt fusion gene. The resulting plasmid, pOG215, was able to express the FC-bDt fusion protein in both Escherichia coli DH5a and S. typhi CVD 908, as evidenced by Western immunoblots using anti-FC and anti-C-terminal diphtheria toxin monoclonal antibodies. Maximum expression of the FC-bDt fusion protein was achieved by growing CVD 908(pOG215) at the low oxidation-reduction potential of thioglycollate broth, i.e. in conditions that activate nirBP and drive transcription of the FC-bDt fusion gene. Whereas maximum expression of FC alone was also observed using thioglycollate broth, expression of bDt alone was unsuccessful using a variety of growth conditions. FC fusions constitute one strategy to "rescue" expression of proteins which are otherwise difficult to express.

The gene encoding the Leishmania major (L. major) promastigote surface glycoprotein, gp63, was introduced into the Salmonella typhimurium (S. typhimurium) aroA- aroD- live oral vaccine strain BRD509 and expressed under the control of a constitutive tac promoter in plasmid pKK233-2. This construct (GID101) expressed gp63 in vitro and was used to immunize highly susceptible BALB/c mice by the oral route. The plasmid was relatively stably inherited by bacteria growing or persisting in the mesenteric lymph nodes of immunized mice. Mice immunized with GID101 developed significant resistance against a challenge infection with L. major compared to controls immunized with BRD509 alone. Spleen and lymph node cells from immunized mice developed a strong in vitro proliferative T-cell response to killed or live L. major. The activated T cells secreted interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) which was abrogated by treatment with anti-CD4 but not with anti-CD8 antibody. The cells did not produce detectable levels of interleukin-4 (IL-4). The immunized mice also produced significant amounts of leishmanial specific IgG2a antibody but did not develop delayed-type hypersensitivity (DTH) to live parasites. No IgG1 antibody was detected. These data therefore demonstrate that gp63 gene delivered orally by a vaccine strain of S. typhimurium can preferentially induce the development of Th-1 subset of CD4+ T cells and protective immunity in the highly susceptible BALB/c mice.

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.

We have not yet directly examined the Th cell responses induced by using Salmonella/BRD 847 as a vector nor have we performed these experiments following immunization with microspheres. However, production of high serum levels of antigen-specific IgG1 may be indicative of a Th2-type response, whereas high serum levels of IgG2a may reflect a Th1-type response. An important issue in using various oral delivery systems is whether the system(s) employed affects the Th cell response to the same antigen. We therefore analyzed the serum antigen-specific IgG subclasses induced in each of the model systems we studied. Table 2 presents these results. Clearly, oral administration of soluble TT with CT as an adjuvant induced an IgG1 subclass, and encapsulation of TT within microspheres had no effect on this Th2-type response. On the other hand, oral immunization with live Salmonella expressing fragment C of tetanus toxin induced a strong IgG2a subclass response indicative of a Th1-type response. It should be noted that protection against a lethal TT challenge was afforded by elevated levels of both anti-TT IgG1 and IgG2a subclasses. We intend to examine the cytokine profiles in spleen CD4+ T cells from mice immunized with microspheres or Salmonella following in vitro antigen stimulation to confirm the T helper type responses suggested by the IgG subclass data. It will also be important to examine the cytokine patterns induced in Peyer's patch CD4+ T cells following immunization of C57BL/6 with Salmonella/BRD 847. Whereas analysis of the serum IgG subclass profile indicated a strong IgG2a response and thus a systemic Th1-type pattern, this vector also induced a good mucosal IgA response. If our current hypothesis concerning S-IgA production is correct, we would expect a predominant Th2-type profile in CD4+ T cells from Peyer's patch in these mice. Such a result would emphasize the bifurcation of T-cell responses in the systemic versus the mucosal immune environments. The data obtained to data suggest that adjuvants and various vehicle delivery systems may influence the induction of distinct T helper cell subsets to a specific antigen. The unique cytokine arrays produced by these T-cell subsets influence the immune responses in terms of systemic Ig subclasses produced, cell-mediated immune responses, and the production of mucosal S-IgA antibodies. Although additional studies are necessary, the manipulation of T-cell subsets employing adjuvants, antigen packaging, or perhaps even the addition of individual cytokines to various formulations holds significant promise for optimizing immune responses to orally administered vaccines.

A TnphoA-generated mutant C5060, attenuated for virulence, was derived from the mouse-virulent Salmonella typhimurium strain C5. This mutation, designated hns-112::TnphoA, harbours the transposon in the 3' end of hns, with the alkaline phosphatase open reading frame in the opposite orientation to that of hns. Bacterial strains harbouring hns-112::TnphoA were mucoid and had altered levels of DNA supercoiling, as monitored using pUC18 as a reporter plasmid. Transduction of hns-112::TnphoA into mouse virulent strains, including S. typhimurium SL1344 and Salmonella enteritidis Se795, resulted in attenuation. When an independent hns mutation, harbouring a kanamycin-resistance cassette inserted into the Kpnl site at base pair 237 of the hns gene, was introduced into S. typhimurium C5, the isolates were also attenuated. S. typhimurium C5 isolates harbouring the multicopy plasmid pGB651, which encodes the Escherichia coli hns gene, were partially attenuated in mice. Transductional analysis, using Tn10 insertions located close to the hns gene, showed that virulence could be restored in genetic crosses that eliminated the resident hns mutations. However, some hns+ transductants were still attenuated, suggesting that secondary attenuating lesions can accumulate in hns-deficient strains. These studies show that the hns locus plays a role in Salmonella virulence.

The use of hepatitis B core antigen (HBcAg) as an immunogenic delivery vehicle for foreign epitopes has been reported. Here we report the insertion of DNA sequences encoding immunodominant linear B-epitopes and a "universal" T-helper epitope of human papillomavirus (HPV) type 16 E7 transforming protein into the full-length HBcAg gene cloned into an inducible bacterial expression plasmid (pPN1.0). The resulting chimeric proteins assembled into particles which were highly immunogenic. Mice immunised with particles containing one or two of the three E7 B-epitopes under consideration produced strong epitope-specific antibody responses with both IgG and IgG2a components which recognised eukaryotic E7. T-proliferative responses were elicited to the E7 T-epitope as well as HBcAg T-epitope(s). Lymph node cells from immunised mice produced IL-2 and IL-4 when specifically recalled in vitro, indicating stimulation of both Th1 and Th2 helper cell compartments. Since HBcAg particles can be administered in adjuvant acceptable for human application and can elicit mucosal responses after nasal or oral immunisation, these results have implications for vaccine design in HPV 16-associated anogenital cancer.

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.

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.

Plasmid pTETnir15, which directs the expression of the non-toxic immunogenic fragment C of tetanus toxin from the anaerobically inducible nirB promoter, was introduced into the Salmonella typhimurium aroA aroD live oral vaccine strain BRD509. The resulting strain, designated BRD847, was used to vaccinate orally BALB/c mice and was tested for plasmid stability and its ability to protect against a lethal tetanus toxin challenge. pTETnir15 was stably inherited by bacteria growing or persisting in the tissues of immunized mice whereas another BRD509 derivative, designated BRD753, harboring plasmid pTET85 which directs fragment C expression from the tac promoter, was highly unstable. Mice immunized with a single oral dose of BRD847 developed high levels of circulating anti-fragment C antibodies and were solidly protected against tetanus toxin challenge. Mice immunized with a single oral dose of BRD753 developed no detectable anti-fragment C antibodies. After boosting, antibodies were detected, but the mice were only partially protected against tetanus toxin challenge. Thus the use of an in vivo inducible promoter such as nirB may be a generally applicable approach to obtaining the stable in vivo expression of heterologous antigens in Salmonella vaccine strains.

We have described a new class of live attenuated salmonella vaccines harbouring lesions in htrA, a stress protein gene previously. The virulence and invasiveness of Salmonella htrA mutants was investigated in three models of increased susceptibility to Salmonella infection. These included BALB/c mice, either given sublethal whole body irradiation (350 R) or administered rabbit anti-TNF alpha antiserum, and (CBA/NfemaleXBALB/cmale)F1 male mice which express the xid sex-linked B cell defect of CBA/N mice and are more susceptible to salmonellae than female littermates. Salmonella typhimurium htrA mutants derived from virulent strains, C5046 (C5 htrA::TnphoA) and BRD726 (SL1344 delta htrA) were not more invasive in immunosuppressed mice than in normal controls in the three mouse models of defective immunity. The results indicate that susceptibility to S. typhimurium htrA vaccines derived from virulent parents is not enhanced by conditions of impaired resistance to infection.

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.

The construction of a Salmonella typhi Ty2 strain harbouring defined deletions in both the aroA and aroC genes is described. These deletions have been fully defined at the molecular level by DNA sequencing and have been introduced in such a way that no foreign DNA remains in the S. typhi genome. This strain is attenuated in mice when given by the intraperitoneal route suspended in hog gastric mucin and is attenuated to a similar level to strains harbouring deletions in aroA or aroC alone indicating that both lesions are capable of attenuating independently. We have used this defined S. typhi aroA aroC strain to express stably a non-toxic 50 kDa fragment of tetanus toxin (fragment C) from a gene incorporated into the chromosome. This strain has the advantage of harbouring no antibiotic-resistance markers and we consider it to be a candidate bivalent oral typhoid-tetanus vaccine.

A mutant of Salmonella choleraesuis was identified that could invade (enter) and penetrate through polarized monolayers of Caco-2 and MDCK epithelial cells at normal levels but was defective for intracellular multiplication within these cells. It was also able to survive inside cultured J774 macrophage cells. These bacteria remained inside membrane-bound vacuoles, which coalesced at later times in the perinuclear region of the epithelial cell. This mutant exhibited slightly slower growth rates in rich or minimal media than the parental strain but was normal for iron usage, phosphate usage, and anaerobic growth and was a prototroph. The mutant was completely avirulent when administered orally or intravenously to susceptible mice. These results suggest that the ability to multiply within eukaryotic cells may contribute to S. choleraesuis virulence.

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.

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.