Jim Hackett

A chromosomal integration system for stabilization of heterologous genes in Salmonella based vaccine strains

We have developed a system whereby heterologous DNA encoding an antigen from an enteropathogen may be recombined into the chromosome of an attenuated Salmonella carrier strain. The system involves two steps: (i) integration of a hisOG deletion mutation into the chromosome; (ii) replacement of the hisOG deletion by the complete hisOG region and the segment of heterologous DNA which encodes the antigen of interest. Recombinants may be selected (his+). The system was used to integrate the genes encoding K88 fimbriae from enterotoxigenic Escherichia coli into the chromosome of a galE mutant of Salmonella typhimurium (LT2H1). Recombinants were detected at a frequency of between 1.0 x 10(-3) and 1.5 x 10(-3). A variety of tests confirmed that the K88 genes were integrated into the chromosome of LT2H1 and were expressed. The stability of the recombinant was tested both in vivo and in vitro. When administered orally to mice, the recombinant elicited a serum antibody response to K88, and retained the Salmonella vaccine potential of the vector strain.

Regulation of the pho regulon of Escherichia coli K-12: Cloning of the regulatory genes phoB and phoR and identification of their gene products☆

Abstract The regulatory genes phoB and phoR of the Escherichia coli K-12 pho regulon have been cloned using two types of selection. The genes were localized on one of the hybrid plasmids, pJP50, by analysis of mutant plasmids generated by Tn5 insertions. Restriction mapping of the other plasmid, pPR20, indicated that the gene order in this region of the chromosome is phoB phoR tsx . Analysis of plasmid-coded proteins in a minicell system showed that: (1) the products of phoB and phoR are proteins with apparent molecular weights of 30,000 and 47,000, respectively; and (2) the synthesis of the phoB gene product is derepressed by phoR mutations. The implications of this observation for the model of pho regulation are discussed.

Production of excreted human epidermal growth factor (hEGF) by an efficient recombinant Escherichia coli system

Recombinant Escherichia coli JM101 strains harbouring plasmids pWKW2 or lacUV5par8EGF, both encoding human epidermal growth factor (hEGF), were used in fermentations to optimize levels of excreted hEGF. Medium composition, inducer level, growth stage at induction and culture conditions, were optimized with respect to volumetric production of the recombinant protein. MMBL medium, with glucose at 5 g:l and tryptone as nitrogen source, was chosen. Isopropyl-b-D-thiogalactopyranoside(IPTG) concentrations of 0.1 mM for E. coli JM101[pWKW2] and 0.2 mM for E. coli K-12 JM101[lacUV5par8EGF], were found to give the best hEGF production levels. The volumetric yields of hEGF were maximal when the cultures were induced in the mid-logarithmic phase. Growth temperature had a significant effect on hEGF yield. A simple continuous fed-batch process for cultivation of E. coli JM101[pWKW2] was developed. The maximum concentration of excreted hEGF attained in continuous fed-batch cultivation was 325 mg:l, as compared to 175 mg:l, in batch cultivation. The hEGF produced from the continuous fed-batch cultivation was substantiated by SDS-PAGE and immunoblotting.

Salmonella-based vaccines.

There continues to be considerable interest in the development of a safe, effective, live, oral vaccine to combat typhoid fever of humans. Such a vaccine may be a derivative of the causative agent of the disease, Salmonella typhi. The prototype of such a vaccine, Ty21a, is not ideal, but no replacement for Ty21a is yet obvious. The construction and trial of bivalent vaccines, in which an attenuated Salmonella strain expresses determinants from another pathogen, awaits the development of a suitably attenuated derivative. In parallel with vaccine development programmes, a variety of techniques have been designed to effect stable association between Salmonella carrier and introduced cloned DNA.

Construction of K88- and K99-expressing clones of Salmonella typhimurium G30: immunogenicity following oral administration to pigs

Salmonella typhimurium G30 was used as a vector to express the ETEC (enterotoxigenic Escherichia coli) fimbrial antigens K88 and K99. Two plasmids encoding K88 or K99 production and having a non-antibiotic selection marker (thyA+) were constructed. These were introduced into a thyA G30 derivative to give the vaccine strains EX841 and EX603, which were shown to express surface K88 or K99, respectively. When administered orally to adult pigs, a dose of 10(11) vaccine organisms elicited significant serum antibody responses to the respective fimbrial antigens. Two such immunizations with EX841 generated serum antibody levels comparable to those obtained with intramuscular injection of killed organisms. Attenuated salmonellae can thus be used to deliver ETEC fimbrial antigens to the porcine intestinal immune system.

A physical map of the chromosomal region determining O-antigen biosynthesis in Vibrio cholerae 01

We have previously described the cosmid cloning of the genes determining the biosynthesis of the Inaba and Ogawa O-antigens of the lipopolysaccharides of Vibrio cholerae O1 (Manning et al., 1986). By Southern hybridization analysis of chromosomal and cosmid DNA, and heteroduplex analysis between the clones we have been able to precisely define the region of contiguous chromosomal DNA in the vicinity of the O-antigen-encoding region. These data and comparison of end points of clones and of deletion derivatives demonstrate that at least 16 kb of a 19-kb SstI fragment is required to encode O-antigen biosynthesis. Expression of O-antigen is independent of the orientation of this SstI fragment with respect to cloning vectors suggesting that its regulatory region has been cloned intact. No detectable differences were observed in the restriction patterns of the Inaba and Ogawa coding regions implying that only minor changes are involved when serotype conversion (Inaba to Ogawa or vice versa) occurs. Bhaskaran [Ind. J. Med. Res. 47 (1959) 253-260] originally defined this region associated with O-antigen biosynthesis oag; however, to be consistent with other organisms [Hitchcock et al., J. Bacteriol. 166 (1986) 699-705], it is suggested this be changed to rfb.

Use of Salmonella for heterologous gene expression and vaccine delivery systems

In the past year, the importance of secretory IgA has been emphasized as fundamental to protection against oral Salmonella infection. In several human trials, aro mutants of Salmonella typhi were highly immunogenic, but still retained the capacity to proceed beyond the gut wall after ingestion. Epitopes of Shiga toxin and influenza hemagglutinin have been expressed in Salmonella surface proteins in work aimed at the construction of hybrid vaccines. Eukaryotic cell involvement in the process of Salmonella attachment/invasion appears to be triggered by host cell phospholipase activation. Our understanding of the number and functions of Salmonella genes involved in the attachment/invasion process has increased considerably--different gene sets are required for invasion of different cell types.

Towards a live oral vaccine against enterotoxigenic Escherichia coli of swine

A live oral vaccine has been developed against scouring induced in piglets by enterotoxigenic Escherichia coli (ETEC). An attenuated strain of Salmonella typhimurium, G30, has been used as a vector for plasmids encoding the production of the fimbrial colonization factors of porcine ETEC. Initial studies with clones expressing K88 or K99 fimbriae have shown them to be well tolerated when administered orally in very high doses. The clones elicited serum, colostral and milk antibodies to the fimbrial antigens, and a challenge trial indicated that such responses were sufficient to ensure the passive transfer of protective immunity to suckling piglets. The possible advantages of this approach are discussed.

Molecular cloning and analysis of the incompatibility and partition functions of the virulence plasmid of Salmonella typhimurium

The incompatibility functions (inc) of the virulence plasmid of Salmonella typhimurium LT2 were initially located in a 4.3 kb region near the repA locus of the plasmid. Expression of inc required the presence, in cis or in trans, of two distinct DNA regions of the fragment. These regions, maximally 0.3 kb and 0.6 kb in size, were separated in the fragment by c. 3.0 kb. This intervening DNA encoded two proteins, of Mr values 37 kDa and 40 kDa. The promoter for the 37 kDa protein lay in or near one of the inc regions (incL). No function was assigned to this protein, however, it may be the product of a rep gene. The 40 kDa protein may have a partition (par) function, and may bind to a centromeric site in or near the other inc region (incR). An inc+ par- derivative of the original plasmid clone was used in a simple method, not involving the use of curing agents/mutagens, to eliminate virulence plasmid DNA from Salmonella typhimurium, Salmonella dublin, Salmonella enteritidis, and Salmonella cholerae-suis. The par function served to stabilise pJRD158B-based plasmid greater than 10(6)-fold in Escherichia coli and the virulence plasmid-cured Salmonella strains.

Extracellular expression of human epidermal growth factor encoded by an Escherichia coli K-12 plasmid stabilized by the ytl2-incR system of Salmonella typhimurium

A plasmid stabilization system, active in high copy-number plasmids, was cloned from the large resident plasmid, pSLT, of Salmonella typhimurium. The ytl2 gene, together with a 249-bp region (termed incR) downstream of the gene, imparted >104-fold stability to a pBR322-based plasmid. The ytl2-incR region was then used to stabilize a recombinant plasmid carrying the human epidermal growth factor gene (with the Escherichia coli K-12 ompA signal sequence), behind the lacUV5 promoter. In shake flask tests to optimize expression of human epidermal growth factor, loss of recombinant plasmid was <1% when growth (both before and after induction with isopropyl-β-d-galactopyranoside) took place even in the absence of antibiotic selection, and the specific activity of secreted human epidermal growth factor was ca 20 μg per 108 cells at harvest, compared to a figure of ca 3 μg per 108 cells when a comparable plasmid, but devoid of the ytl2-incR region, was employed, as outgrowth of plasmid-free cells after induction severely compromised the specific activity of the secreted product.