Mitomycin C induction of bacteriophages from Serpulina hyodysenteriae and Serpulina innocens

Abstract 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.     

A single dose of recombinant Salmonella typhimurium induces specific humoral immune responses against heterologous Eimeria tenella antigens in chicken

Salmonella typhimurium vaccine strains were used as antigen delivery system for oral immunisation of chickens against two antigens of the coccidian parasite Eimeria tenella. The cDNAs of the known E. tenella proteins, SO7 and TA4, were isolated from total RNA and subcloned into the expression vectors pQE30 and pTECH2. Subcutaneous immunisation of chickens with Escherichia coli-expressed SO7 and TA4 revealed that both proteins were immunogenic. Both cDNAs were subcloned into plasmids of the pTECH2 vector system, which allows them to be expressed as fusion proteins with the highly immunogenic fragment C of the tetanus toxin under control of the anaerobically inducible nirB promoter. Plasmids were introduced into the S. typhimurium vaccine strains SL3261, C5aroD and C5htrA. SDS-PAGE and Western blot analysis revealed expression of both fusion proteins in all strains under anaerobic culture conditions. Three-week-old white leghorn chickens were orally immunised with 10(9) CFU per animal. The stability of the recombinant bacteria was revealed by recovery of viable Salmonella containing the respective plasmids from the liver of the immunised chickens at day 3 after inoculation. Specific serum IgG antibodies against the SO7-or TA4-antigens were detectable by ELISA 2 weeks after oral immunisation and remained for at least 6 weeks, while specific IgA antibodies were restricted to the bile of the birds. All chickens produced serum IgG and IgA to S. typhimurium lipopolysaccharides. Our data show that a single oral inoculation with recombinant S. typhimurium SL3261, C5aroD and C5htrA can induce specific antibody responses to heterologous Eimeria antigens in chickens, suggesting that recombinant Salmonella are a suitable delivery system for vaccines against Eimeria infections.     

Antigen delivery by attenuated Bacillus anthracis: new prospects in veterinary vaccines

This report summarizes the recent investigations on the use of Bacillus anthracis as a live vector for delivery of antigens. Recombinant strains were constructed by engineering the current live Sterne vaccine. This vaccine, used to prevent anthrax in cattle, causes side-effects due to anthrax toxin activities. Bacteria producing a genetically detoxified toxin factor were devoid of lethal effects and were as protective as the Sterne strain against experimental anthrax. Moreover, B. anthracis expressing a foreign antigen controlled by an in vivo inducible promoter were able to generate either antibody or cellular protective responses against heterologous diseases.     

Attenuated Salmonella enterica serovar typhi live vector with inducible chromosomal expression of the T7 RNA polymerase and its evaluation with reporter genes

Attenuated Salmonella strains with defined gene deletions have been extensively evaluated as suitable live carriers of passenger antigens. A number of strategies for antigen delivery by these strains have been attempted, ranging from plasmid-based to chromosomal integration systems. We report here the chromosomal integration of the T7 RNA polymerase gene (T7pol) in the attenuated strain Salmonella enterica serovar Typhi (Salmonella typhi) CVD908 (aroC(-), aroD(-)). The T7pol gene was amplified by PCR from Escherichia coli BL21(DE3) and cloned in the pNir3 plasmid under the control of the anaerobically inducible nirB promoter. Then it was subcloned in a pKTN701 derivative, suicide plasmid with the R6K ori, and flanked by the aroC gene. After evaluation of its functionality in E. coli SY327, the aroC-T7pol-aroC cassette was integrated into the aroC locus of S. typhi CVD908 by homologous recombination. The resulting strain, S. typhi CVD908-T7pol, was able to transcomplement two plasmids bearing the luc or the lacZ reporter genes controlled by the T7 promoter and produce luciferase and beta-galactosidase under anaerobic culture conditions. Therefore, an inducible system for recombinant antigen production in attenuated S. typhi was achieved.     

Use of stabilized luciferase-expressing plasmids to examine in vivo-induced promoters in the Vibrio cholerae vaccine strain CVD 103-HgR

Live, attenuated Vibrio cholerae vaccines can induce potent immune responses after only a single oral dose. The strategy of harnessing these strains to present antigens from heterologous pathogens to the mucosal immune system shows great promise. To fully realize this possibility, V. cholerae strains must be created that stably express antigens in vivo in sufficient quantity to generate an immune response. In vivo -induced promoters have been shown to increase the stability and immunogenicity of foreign antigens expressed from multicopy plasmids. We report the construction of a series of genetically stabilized plasmids expressing luciferase as a heterologous protein from the following in vivo -induced promoters: V. cholerae P _argC , P _fhuC and P _vca1008 , and Salmonella enterica serovar Typhi P _ompC . We demonstrate that several of these expression plasmids meet two critical criteria for V. cholerae live vector vaccine studies. First, the plasmids are highly stable in the V. cholerae vaccine strain CVD 103-HgR at low copy number, in the absence of selective pressure. Second, real-time bioluminescent imaging (BLI) demonstrates inducible in vivo expression of the promoters in the suckling mouse model of V. cholerae colonization. Moreover, the use of BLI allows for direct quantitative comparison of in vivo expression from four different promoters at various time points.     

Enzyme production by recombinant Trichoderma reesei strains.

The production of both homologous and heterologous proteins with the cellulolytic filamentous fungus Trichoderma reesei is described. Biotechnically important improvements in the production of cellulolytic enzymes have been obtained by genetic engineering methodology to construct strains secreting novel mixtures of cellulases. These improvements have been achieved by gene inactivation and promoter changes. The strong and highly inducible promoter of the gene encoding the major cellulase, cellobiohydrolase I (CBHI) has also been used for the production of eukaryotic heterologous proteins in Trichoderma. The expression and secretion of active calf chymosin is described in detail.     

High level heterologous expression in E. coli using the anaerobically-activated nirB promoter.

The anaerobically-regulated nirB promoter was used to express heterologous genes in Escherichia coli. Under anaerobic conditions the promoter was able to express tetanus toxin fragment C at approximately 20% total cell protein (tcp) and the Bordetella pertussis antigen pertactin at greater than 30% tcp. These levels are comparable to those obtained for the same products using the tac promoter. The nirB promoter is very well regulated, giving almost two orders of magnitude increase in fragment C on complete removal of oxygen. The use of this anaerobically-induced promoter in the production of recombinant proteins in E. coli is discussed.     

The development of oral vaccines based on live attenuated Salmonella strains

Abstract 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.     

Secretory delivery of recombinant proteins in attenuated Salmonella strains: potential and limitations of Type I protein transporters

Live attenuated Salmonella strains have been extensively explored as oral delivery systems for recombinant vaccine antigens and effector proteins with immunoadjuvant and immunomodulatory potential. The feasibility of this approach was demonstrated in human vaccination trials for various antigens. However, immunization efficiencies with live vaccines are generally significantly lower compared to those monitored in parenteral immunizations with the same vaccine antigen. This is, at least partly, due to the lack of secretory expression systems, enabling large-scale extracellular delivery of vaccine and effector proteins by these strains. Because of their low complexity and the terminal location of the secretion signal in the secreted protein, Type I (ATP-binding cassette) secretion systems appear to be particularly suited for development of such recombinant extracellular expression systems. So far, the Escherichia coli hemolysin system is the only Type I secretion system, which has been adapted to recombinant protein secretion in Salmonella. However, this system has a number of disadvantages, including low secretion capacity, complex genetic regulation, and structural restriction to the secreted protein, which eventually hinder high-level in vivo delivery of recombinant vaccines and effector proteins. Thus, the development of more efficient recombinant protein secretion systems, based on Type I exporters can help to improve efficacies of live recombinant Salmonella vaccines. Type I secretion systems, mediating secretion of bacterial surface layer proteins, such as RsaA in Caulobacter crescentus, are discussed as promising candidates for improved secretory delivery systems.     

A method to generate recombinant <Emphasis Type="Italic">Salmonella typhi</Emphasis> Ty21a strains expressing multiple heterologous genes using an improved recombineering strategy

Live attenuated Salmonella enterica serovar Typhi Ty21a (Ty21a) is an important vaccine strain used in clinical studies for typhoid fever and as a vaccine vector for the expression of heterologous antigens. To facilitate the use of Ty21a in such studies, it is desirable to develop improved strategies that enable the stable chromosomal integration and expression of multiple heterologous antigens. The phage λ Red homologous recombination system has previously been used in various gram-negative bacteria species to mediate the accurate replacement of regions of chromosomal DNA with PCR-generated ‘targeting cassettes’ that contain flanking regions of shared homologous DNA sequence. However, the efficiency of λ Red-mediated recombineering in Ty21a is far lower than in Escherichia coli and other Salmonella typhimurium strains. Here, we describe an improved strategy for recombineering-based methods in Ty21a. Our reliable and efficient method involves the use of linear DNA-targeting cassettes that contain relatively long flanking ‘arms’ of sequence (ca. 1,000 bp) homologous to the chromosomal target. This enables multiple gene-targeting procedures to be performed on a single Ty21a chromosome in a straightforward, sequential manner. Using this strategy, we inserted three different influenza antigen expression cassettes as well as a green fluorescent protein gene reporter into four different loci on the Ty21a chromosome, with high efficiency and accuracy. Fluorescent microscopy and Western blotting analysis confirmed that strong inducible expression of all four heterologous genes could be achieved. In summary, we have developed an efficient, robust, and versatile method that may be used to construct recombinant Ty21a antigen-expressing strains.     

Use of recombinant Salmonella strains for supplying the macro-organism with biologically active molecules

The characterization of known Salmonella vaccine strains and different attenuated mutants used for developing new vaccines is presented. The use of attenuated Salmonella strains as vaccine vector for the supply of heterologous antigens opens prospects for the creation of effective and commonly available vaccines which approximate the "ideal" vaccine in their qualitative characteristics. The possibility of the genetic modification of attenuated strains permits their targeted reconstruction, considering the specific features of the formation of immune response to the definite heterologous antigen supplied to the body by the bacterial vector.     

Novel bacterial systems for the delivery of recombinant protein or DNA

On the basis of attenuated intracellular bacteria, we have developed two delivery systems for either heterologous proteins or DNA vaccine vectors. The first system utilizes attenuated strains of Gram-negative bacteria which are engineered to secrete heterologous antigens via the alpha-hemolysin secretion system of Escherichia coli. The second system is based on attenuated suicide strains of Listeria monocytogenes, which are used for the direct delivery of eukaryotic antigen expression vectors into professional antigen presenting cells (APC) like macrophages in vitro as well as in vivo.     

A dps promoter based expression system for improved solubility of expressed proteins in Escherichia coli

Escherichia coli is widely used for recombinant protein production due to its well established genetic manipulation techniques and cost effectiveness of the associated production processes. Soluble expression of heterologous recombinant proteins constitutes a major problem in the deployment of bacterial expression systems. We have developed a dps promoter based expression system in E. coli for improved solubility of expressed proteins. The resulting expression system was found to be superior to the IPTG inducible T7 promoter based pET expression system for production of soluble β-galactosidase, tdTomato, and mCherry. The dps promoter based expression system was shown to be functional in most commonly used strains of E. coli without need for prior genetic manipulation of the host genome.     

Delivery of a heterologous antigen by a registered Salmonella vaccine (STM1)

STM1 is an aro A(-) attenuated mutant of Salmonella enterica serovar Typhimurium, and is a well-characterised vaccine strain available to the livestock industry for the prevention of salmonellosis in chickens. This strain has potential for heterologous antigen delivery, and here we show that the strain can be used to deliver a model antigen, ovalbumin, to immune cells in vitro and in vivo. Two plasmid constructs expressing the ovalbumin gene were utilised, one of which uses a prokaryotic promoter and the other the CMV promoter (DNA vaccine). In vitro, STM1 carrying ovalbumin-encoding plasmids was able to invade dendritic cells and stimulate a CD8(+) cell line specific for the dominant ovalbumin epitope, SIINFEKL. In vivo, spleen cells were responsive to SIINFEKL after vaccination of mice with ovalbumin-encoding plasmids in STM1, and finally, humoral responses, including IgA, were induced after vaccination.     

Rational design of Salmonella-based vaccination strategies

A permanently growing body of information is becoming available about the quality of protective immune responses induced by mucosal immunization. Attenuated live bacterial vaccines can be administered orally and induce long-lasting protective immunity in humans without causing major side effects. An attenuated Salmonella enterica serovar Typhi strain is registered as live oral vaccine against typhoid fever and has been in use for more than two decades. Recombinant attenuated Salmonella strains are also an attractive means of delivering heterologous antigens to the immune system, thereby, stimulating strong mucosal and systemic immune responses and consequently provide an efficient platform technology to design novel vaccination strategies. This includes the choice of heterologous protective antigens and their expression under the control of appropriate promoters within the carrier strain. The availability of well-characterized attenuated mutants of Salmonella concomitantly supports fine tuning of immune response triggered against heterologous antigens. Exploring different mucosal sites as a potential route of immunization has to be taken into account as an additional important way to modulate immune responses according to clinical requirements. This article focuses on the rational design of strategies to modulate appropriate immunological effector functions on the basis of selection of (i) attenuating mutations of the Salmonella strains, (ii) specific expression systems for the heterologous antigens, and (iii) route of mucosal administration.     

Use of a transactive regulatory mutant of Dictyostelium discoideum in a eucaryotic expression system.

The discoidin proteins of Dictyostelium discoideum are highly expressed during development. The Disc I gamma promoter allows the regulation of heterologous protein expression by experimental conditions. We report conditions under which the promoter activity is efficiently repressed during growth in the wildtype strain AX2. In addition we show that a mutant which overexpresses the discoidins also overexpresses the reporter genes beta-galactosidase, luciferase and CAT 10- to 100-fold when these are placed under the control of a Disc I gamma promoter. This system may be generally useful for the overexpression of genes in Dictyostelium, both for functional studies in vivo and for the production of heterologous proteins for purification.     

Molecular genetic analysis of an FNR-dependent anaerobically inducible Escherichia coli promoter

From the effects of 13 deletions and three linker-scanner mutations at the Escherichia coli nirB promoter we have located sequences necessary for FNR-dependent induction of activity by anaerobiosis and further nitrite-dependent stimulation of expression. We describe a nirB promoter derivative that allows the cloning of 'cassettes' carrying different FNR-binding sequences and experiments in which a number of point mutations were introduced into these sequences. FNR-dependent stimulation of expression from the nirB promoter is critically dependent on the location of the FNR-binding site, and deletion or insertion of one base pair is sufficient to disrupt promoter function. We have transferred a number of cassette FNR-binding sequences from the nirB promoter to the unrelated melR promoter. The insertion of FNR-binding sequences at the melR promoter is sufficient to confer fnr-dependency on expression. However expression from these hybrid promoters is not as efficiently repressed during aerobic growth, suggesting that the function of bound FNR is dependent on the sequence context of the FNR-binding sequence.     

Bacterial virulence, proinflammatory cytokines and host immunity: how to choose the appropriate Salmonella vaccine strain?

Salmonella infection in its mammalian host can be dissected into two main components. The co-ordinate expression of bacterial virulence genes which are designed to evade, subvert or circumvent the host response on the one hand, and the host defence mechanisms which are designed to restrict bacterial survival and replication on the other hand. The outcome of infection is determined by the one which succeeds in disturbing this equilibrium more efficiently. This delicate balance between Salmonella virulence and host immunity/inflammation has important implications for vaccine development or therapeutic intervention. Novel Salmonella vaccine candidates and live carriers for heterologous antigens are attenuated strains with defined genetic modifications of metabolic or virulence functions. Although genetic defects of different gene loci can lead to similar degrees of attenuation, effects on the course of infection may vary, thereby altering the quality of the elicited immune response. Studies with gene-deficient animals indicate that Salmonella typhimurium strains with mutations in aroA, phoP/phoQ or ssrA/ssrB invoke different immune responses and that a differential repertoire of pro-inflammatory cytokines is required for clearance. Consequently, Salmonella mutants defective in distinct virulence functions offer the potential to specifically modulate the immune response for defined medical applications.