Bruce A. D. Stocker

Effect of different purine auxotrophic mutations on mouse-virulence of a Vi-positive strain of Salmonella dublin and of two strains of Salmonella typhimurium☆

Ten cfu of a Vi-positive strain of Salmonella dublin, given i.p. to BALB/c mice, caused death. Of transductional derivatives with nutritional requirements expected to reduce virulence, those with complete blocks at aroA (therefore aromatic-dependent) or at purA (therefore adenine-dependent) were non-virulent; no deaths from i.p. inocula of 10(6) or 10(7) cfu, respectively. Transductants with identified blocks at purF, purG, purC or in the purJHD operon, all with purine requirement satisfied by hypoxanthine or any other purine, were of reduced virulence, with some deaths from 10(4) cfu or, for most of the derivatives, from only 10(2) cfu. Blocks at guaA or guaB, causing guanine requirement, likewise resulted only in reduced virulence, with some deaths from inocula of 10(2). Transductional derivatives of two mouse-virulent (LD50, i.p., less than 25 cfu) strains of S. typhimurium were similarly tested, with generally similar results, in that purA and purB (adenine-requiring) transductants were non-virulent, no deaths from 2.5 x 10(7) cfu, and those with purF, purG or purC defects (hypoxanthine-responding) retained partial virulence. However, the guaA and guaB (guanine-requiring) derivatives were of much reduced virulence; all mice survived 2.5 x 10(5) cfu but only one survived 2.5 x 10(7) cfu. In both the S. dublin and the S. typhimurium tests autopsy cultures showed that fatal infections resulted from multiplication of the auxotrophic strain, not from that of non-exacting revertants.

Auxotrophic Salmonella typhi as live vaccine

Salmonella typhi 541Ty has deletions at aroA and purA, causing requirement for aromatic metabolites (including p-aminobenzoate) and for adenine. None of 36 volunteers who drank 10(8) to 10(10) bacteria of 541Ty or its Vi-negative mutant 543Ty showed any adverse effect; all gave evidence of cellular immune response but only a few had serum titre increases. S. typhimurium experiments (at the Wellcome Research Laboratories and at Stanford) show that adenine requirement may reduce both bacterial survival in mouse tissues and live-vaccine efficacy. S. typhi attenuated only by block(s) in aromatic biosynthesis may be more effective as oral-route live vaccine.

A key role for type 1 pili in enterobacterial communicability

Up to 80% of faecal Escherichia coil strains are able to produce type 1 pili. These filamentous bacterial surface organelles, which mediate mannose‐sensitive attachment to mammalian epithelial cells, are also conserved throughout the Enterobacteriaceae. As a potential explanation for their prevalence among intestinal isolates of enteric bacteria, it has been widely speculated that type 1 pili are important for adherence to the hosts intestinal mucosa. However, conclusive evidence for this idea is lacking, and there are reasonable grounds for doubting such an effect. Permanent interruption of type 1 pillation in previously pil+E. coli (by directed mutagenesis of pilA, the gene coding for the major structural subunit of type 1 pili) does not diminish the density of intestinal colonization in individual animals. Rather, as we demonstrate here, this lesion results in a dramatic decrease in transmission of E. coli K1 from experimentally colonized neonatal rats to their littermates. The enhanced communicability associated with type 1 pillation suggests a heretofore unrecognized explanation for the prevalence of type 1 pili among intestinal E. coli; one that does not necessarily require the direct action of these organelles at the intestinal mucosa.

Protection against oral challenge three months after i.v. immunization of BALBc mice with live Aro Salmonella typhimurium and Salmonella enteritidis vaccines is serotype (species)-dependent and only partially determined by the main LPS O antigen

The role of the main LPS O antigen in the specificity of protection as mediated by systemic mechanisms following immunization with live attenuated Aro Salmonella vaccines was studied in mice. Innately Salmonella-susceptible (Itys) BALB/c mice were immunized intravenously with a single dose of either Salmonella typhimurium SL3261 aroA (LPS O4,5,12) or Salmonella enteritidis Se795aroA (LPS O1,9,12), and challenged orally 2-3 months later with either S. typhimurium C5 or S. enteritidis Thirsk. Nearly isogenic transductants of the two challenge strains expressing either their own LPS or that of the other serotype (S. typhimurium C5 O4 or O9, and S. enteritidis Thirsk O9 or O4) were also used. Both vaccines conferred similar high protection against the virulent strain of the homologous serotype expressing its own LPS. There was no protection against the heterologous serotype expressing its own LPS. However, when vaccinated mice were challenged with either the same serotype as the vaccine but expressing the heterologous LPS, or with the heterologous serotype expressing the LPS of the vaccine, protection was always lower than protection against the fully homologous serotype. Anti-smooth LPS antibodies showed higher titres against the homologous LPS, but with significant crossreactivity with the heterologous LPS. Antibodies to O-rough S. typhimurium and S. enteritidis LPS were present following immunization with either of the two vaccine strains. The LPS alone cannot fully account for the specificity of protection in this model; other (protein) antigens may be responsible. It remains to be seen whether there is a T-cell mediated component to the specificity of protection conferred by live Salmonella vaccines.

Development of an auxotrophic oral live Shigella flexneri vaccine

An oral live attenuated Shigella flexneri vaccine candidate strain was constructed by making it auxotrophic and dependent on aromatic metabolites not available in mammalian tissues. An aroD gene of Escherichia coli K12 strain NK 5131, inactivated by insertion in it of the Tn 10 transposon, was transduced using phage P1 into a virulent S. flexneri serotype Y strain (Sfl 1) isolated from a patient with bacillary dysentery. One of the transductant strains Sfl 114 was found to invade HeLa cells in vitro, to cause plaque formation in HeLa monolayers (i.e. maintain intracellular multiplication in vitro), but to be unable to cause keratoconjunctivitis in guinea-pig eyes. When the strain was fed to Macacca fascicularis monkeys it was well tolerated, excreted for 1-4 days, and found to elicit a local intestinal sIgA and serum IgA, IgM and IgG responses. Monkeys challenged with 100 ID50 dose (1 X 10(11) bacteria) of the virulent parent Sfl 1 strain were completely protected from development of diarrhoea. Coloscopy of the monkeys and the sampling of intestinal biopsies showed that the vaccine protected against the surface epithelial erosions and ulcerations seen in unimmunized monkeys. Killing of invading virulent shigellae apparently took place intracellularly in the mucosa suggesting that cellular immune mechanisms played a role in the elicited host defence. The constructed S. flexneri Sfl 114 strain has the properties of a promising shigella vaccine and will next be the subject of studies with human volunteers.

Immunity conferred by Aro− salmonella live vaccines

The specificity of protection conferred by Aro- salmonellae was studied in BALB/c mice challenged 3 months after intravenous (i.v.) vaccination, more than 1 month after the vaccine had been cleared. Oral challenge showed better protection than i.v. challenge. Salmonella typhimurium aroA SL3261 conferred very good protection against wild-type S. typhimurium C5 (over 10,000 x LD50). Cross protection experiments were performed using S. typhimurium, S. enteritidis and S. dublin for vaccination and challenge, including variants of S. typhimurium and S. enteritidis of similar virulence differing in the main LPS antigen (O-4 or O-9). Salmonella typhimurium aroA conferred solid protection against S. typhimurium (O-4), but no protection against wild-type S. enteritidis (O-9). However challenge with LPS variant strains showed that although protection was generally better to strains of the homologous LPS type, specificity of protection was determined more by the parent strain background (S. typhimurium or S. enteritidis) of the challenge than by O-factors 4 or 9, suggesting that other antigens are involved. The nature of the protective antigen(s) in this model is unclear, but it does not appear to be the main O-specific antigen. A S. enteritidis Se795 aroA vaccine gave good protection against wild-type S. enteritidis Se795 2 weeks after vaccination, but much less at 3 months (approximately 10-200 x LD50), although the persistence of the S. enteritidis aroA vaccine in the liver and spleen was similar to that of the S. typhimurium vaccine, and the wild-type Se795 challenge strain was of similar virulence to S. typhimurium C5. A S. dublin aroA vaccine conferred similar protection against wild-type S. dublin (approximately 300 x LD50).

Expression and immunogenicity of an 18-residue epitope of HIV1 gp41 inserted in the flagellar protein of a Salmonella live vaccine.

A synthetic oligonucleotide specifying residues 735-752 of the product of the env gene of HIV1 IIIB was inserted by blunt-end ligation at restriction sites in the hypervariable, antigenically determinant region IV of two flagellin genes. Its integration, in frame and correct orientation, into gene fliC(d) in plasmid pLS408 allowed production of functional flagella when the plasmid was placed in a flagellin-negative aroA live-vaccine Salmonella dublin strain, SL5928. Bacteria thus made motile were immobilized and agglutinated by anti-(735-752 peptide) serum; expression was also shown by immunoelectron-microscopy and by Western blot of whole-cell lysates. Enzyme immunoassay (EIA) of sera of mice given three doses by intraperitoneal injection of the live-vaccine strain producing chimeric flagellin, or of concentrated flagella from it, showed production of antibody with affinity for the peptide, and in some sera, also for r-gp160. Pooled serum from mice given five i.p. doses of the live vaccine strain expressing the gp41 epitope at the surface of its flagellar filaments had higher titres of anti-peptide and anti-r-gp160 antibody and weak neutralizing activity on the IIIB isolate (90% neutralization at 1/100). The sera of nine mice given two doses of the live vaccine by the oral route had either no or only very low titres of antibody to flagellar antigen d; they were therefore not tested for anti-peptide activity.

Immune Responses to Epitopes Inserted in Salmonella Flagellin

Plasmid pLS408 includes gene fliC(d) specifying Salmonella flagellin of antigenic type d with an in vitro deletion of a 48 base-pair EcoRV fragment in its central hypervariable antigenically-determinant region IV. Oligonucleotides specifying peptide epitopes of antigens of unrelated pathogens inserted, in correct orientation, at the unique EcoRV site of pLS408 specify chimeric flagellins and, in many instances, cause production of functional flagella when the plasmid is placed in a flagellin-deficient delta aroA live-vaccine strain of Salmonella dublin. The foreign epitope is then exposed at the surface of the flagellar filaments, as shown by the immobilizing effect of anti-epitope antibody and by immunogold electron-microscopy. The live-vaccine strain with a foreign epitope at the surface of its flagella when administered to mice by injection nearly always causes production of antibody with affinity for the foreign epitope and, sometimes, also for the source protein. Repeated injection of the live vaccine with an epitope of Streptococcus pyogenes type 5 M protein as insert caused production of opsonizing antibody and conferred partial protection against Streptococcus challenge. Injection of semi-purified chimeric flagella or flagellin, alone or with adjuvant, likewise causes antibody production, in one instance sufficient to give partial protection against influenza A virus challenge. Plasmid pLS408 with some inserts does not confer motility, either because the filaments produced are non-functional or because flagellin is made but not assembled or because little or no flagellin is produced. The features of a sequence which as insert determine production or non-production of functional flagella are not known. The effect of insertion of known T-cell epitopes and cellular immune responses to epitope inserts in flagellin are as yet little explored.

Delivery of class I and class II MHC-restricted T-cell epitopes of listeriolysin of Listeria monocytogenes by attenuated Salmonella

Using a Salmonella vaccine-Listeria infection model of intracellular infection, we studied the capacity of an attenuated strain of Salmonella carrying T-cell epitopes of listeriolysin (LLO) of L. monocytogenes to elicit epitope-specific T-cell responses. Class II (LLO 215-226) or class I (LLO 91-99) MHC-restricted T-cell epitopes of LLO were inserted within a central, hypervariable domain of the flagellin protein of an attenuated delta aroA Salmonella dublin strain. T cells from Listeria-immunized mice were activated by lysates or heat-killed preparations of Salmonella construct expressing the LLO 215-226 epitope, indicating that LLO 215-226 is processed and presented to T cells when offered to antigen-presenting cells as part of a flagellin-epitope fusion protein. The chimeric flagellin genes were integrated into the chromosome of the flagellin-negative S. dublin strain to obtain stable expression of the epitopes. Immunization with the living, chromosomally integrated Salmonella construct carrying LLO 215-226 epitope as part of the flagellin protein generated T cells reactive with the corresponding LLO peptide, indicating that this chimera can stimulate a class-specific immune response in vitro. The effect of flanking residues on the processing and presentation of MHC class I LLO 91-99 epitope was studied using Salmonella vaccine strains that express chimeric flagellins containing one of three LLO 91-99 inserts: 91-99 (normal flagellin amino acids as flanking residues); KK91-99KK (Lys-Lys flanking residues); and AAA91-99AAA (Ala-Ala-Ala flanking residues).(ABSTRACT TRUNCATED AT 250 WORDS)

Intragenic recombination in a flagellin gene: characterization of the H1-j gene of Salmonella typhi.

Salmonella typhi, the etiologic agent of typhoid fever, typically has only a phase‐1 flagellar antigen, d, but some isolates, found only in Indonesia, have antigen j instead, and may have a second flagellar antigen, z66. It appears that intragenic recombination involving a directly repeated 11 bp sequence in the H1‐d flagellin gene changed the flagellar antigen to j, by deleting 261 bp in its central, antigenically determinant, part. Sequencing of the hypervariable regions of genes H1‐d and H1‐j, and hybridization of such genes, after amplification by the polymerase chain reaction, with oligonucleotide probes specific for the deleted segment or for the sequence produced by the recombination confirmed that all the j alleles have the postulated deletion. By applying the polymerase chain reaction to study S. typhi isolates from Jakarta, not previously tested in respect to flagellar antigen, we showed that gene H1‐j was nearly as common as H1‐d in these isolates.