Molecular characterization of Clostridium perfringens isolates from humans with sporadic diarrhea: evidence for transcriptional regulation of the beta2-toxin-encoding gene.

Clostridium perfringens type A food poisoning is caused by C. perfringens isolates carrying a chromosomal enterotoxin gene (cpe), while non-food-borne gastrointestinal (GI) diseases, such as antibiotic-associated diarrhea (AAD) and sporadic diarrhea (SD), are caused by C. perfringens plasmid cpe isolates. A recent study reported the association of beta2 toxin (CPB2) with human GI diseases, and particularly AAD/SD, by demonstrating that a large percentage of AAD/SD isolates, in contrast to a small percentage of food poisoning isolates, carry the beta2-toxin gene (cpb2). This putative relationship was further tested in the current study by characterizing 14 cpe+ C. perfringens fecal isolates associated with recent cases of human SD in England (referred to hereafter as SD isolates). These SD isolates were all classified as cpe+ type A, and 12 of the 14 cpe+ isolates carry their cpe gene on the plasmid and 2 carry it on the chromosome. Interestingly, cpb2 is present in only 12 plasmid cpe isolates; 11 isolates carry cpe and cpb2 on different plasmids, but cpe and cpb2 are located on the same plasmid in one isolate. C. perfringens enterotoxin is produced by all 14 cpe+ SD isolates. However, only 10 of the 12 cpe+/cpb2+ SD isolates produced CPB2, with significant variation in amounts. The levels of cpb2 mRNA in low- to high-CPB2-producing SD isolates differed to such an extent (30-fold) that this difference could be considered a major cause of the differential level of CPB2 production in vitro by SD isolates. Furthermore, no silent or atypical cpb2 was found in a CPB2 Western blot-negative isolate, 5422/94, suggesting that the lack of CPB2 production in 5422/94 was due to low expression of cpb2 mRNA. This received support from our observation that the recombinant plasmid carrying 5422/94 cpb2, which overexpressed cpb2 mRNA, restored CPB2 production in F4969 (a cpb2-negative isolate). Collectively, our present results suggest that CPB2 merits further study as an accessory toxin in C. perfringens-associated SD.

Regulated expression of the beta2-toxin gene (cpb2) in Clostridium perfringens type a isolates from horses with gastrointestinal diseases.

Recent epidemiological studies suggested that cpb2-positive Clostridium perfringens isolates are associated with gastrointestinal (GI) diseases in horses. These putative relationships, indicated by PCR genotyping, were tested in the present study by further genotyping and phenotyping of 23 cpb2-positive C. perfringens isolates from horses with GI disease (referred to hereafter as horse GI disease isolates). Our beta2-toxin (CPB2) Western blot analyses demonstrated that all of the tested isolates were unable to produce detectable levels of CPB2. However, Southern blot and nucleotide sequencing analyses identified intact cpb2 open reading frames in all of our surveyed horse GI disease isolates. Furthermore, reverse transcriptase PCR and Northern blot analyses showed that cpb2 genes in all of our surveyed horse GI disease isolates were transcriptionally active, i.e., an approximately 1.2-kb cpb2-specific mRNA was identified in total RNA from our surveyed isolates. The levels of cpb2 mRNA in CWC245 (a high-CPB2-producing pig strain) and our surveyed horse GI disease isolates differed to such an extent (35-fold) that this difference could be considered as a major cause of the difference in levels of CPB2 production by CWC245 and horse GI disease isolates. This finding received further support from our observation that the complementing strain 106902(pMRS140), which produced significantly higher levels of mRNA than strain 106902, produced high levels of CPB2. Collectively, our results indicated that there is a positive correlation between cpb2 transcription levels and the amount of CPB2 produced by a C. perfringens cell and that decreased transcription and/or message instability may be involved, at least in part, in the low CPB2 production noted for horse GI disease isolates in comparison to that noted for pig GI disease isolate CWC245.

DNA vaccines for biodefence.

The advantages associated with DNA vaccines include the speed with which they may be constructed and produced at large-scale, the ability to produce a broad spectrum of immune responses, and the ability for delivery using non-invasive means. In addition, DNA vaccines may be manipulated to express multiple antigens and may be tailored for the induction of appropriate immune responses. These advantages make DNA vaccination a promising approach for the development of vaccines for biodefence. In this review, the potential of DNA vaccines for biodefence is discussed.

Evaluation of the VP22 protein for enhancement of a DNA vaccine against anthrax.

BACKGROUND: Previously, antigens expressed from DNA vaccines have been fused to the VP22 protein from Herpes Simplex Virus type I in order to improve efficacy. However, the immune enhancing mechanism of VP22 is poorly understood and initial suggestions that VP22 can mediate intercellular spread have been questioned. Despite this, fusion of VP22 to antigens expressed from DNA vaccines has improved immune responses, particularly to non-secreted antigens. METHODS: In this study, we fused the gene for the VP22 protein to the gene for Protective Antigen (PA) from Bacillus anthracis, the causative agent of anthrax. Protective immunity against infection with B. anthracis is almost entirely based on a response to PA and we have generated two constructs, where VP22 is fused to either the N- or the C-terminus of the 63 kDa protease-cleaved fragment of PA (PA63). RESULTS: Following gene gun immunisation of A/J mice with these constructs, we observed no improvement in the anti-PA antibody response generated. Following an intraperitoneal challenge with 70 50% lethal doses of B. anthracis strain STI spores, no difference in protection was evident in groups immunised with the DNA vaccine expressing PA63 and the DNA vaccines expressing fusion proteins of PA63 with VP22. CONCLUSION: VP22 fusion does not improve the protection of A/J mice against live spore challenge following immunisation of DNA vaccines expressing PA63.

An association between ATP binding cassette systems, genome sizes and lifestyles of bacteria.

Comparative bioinformatic analyses of ATP binding cassette (ABC) systems encoded in bacterial genomes have been undertaken in order to examine whether the range and distribution of these systems correlates with niches occupied by different organisms. In general, bacteria with larger genomes were found to encode more ABC systems than those with smaller genomes. Environmental bacteria, generally containing the largest genomes, showed the greatest number and diversity of ABC systems. Extracellular bacteria have larger genomes and show higher relative numbers of ABC transporters in comparison to intracellular bacteria. Similar results were obtained when comparing bacteria with different respiratory requirements since aerobic bacteria have larger genomes and also display greater numbers of ABC systems than anaerobes. These results suggest that the number of ABC systems encoded in bacterial genomes correlates with genome size and also with the physiological niche in which bacteria live. Furthermore, the distribution of the ABC systems into families indicates that the process of reductive evolution is responsible for retaining particular types of ABC systems as bacteria adapt to particular niches.

Antibiotic-free plasmid stabilization by operator-repressor titration for vaccine delivery by using live Salmonella enterica Serovar typhimurium.

Live, attenuated bacteria are effective vectors for heterologous antigen delivery. However, loss of heterologous gene-bearing plasmids is problematic, and antibiotics and their resistance genes are not desirable for in vivo DNA vaccine delivery due to biosafety and regulatory concerns. To solve this problem, we engineered the first vaccine delivery strain that has no requirement for antibiotics or other selectable marker genes to maintain the recombinant plasmid. This model strain of Salmonella enterica serovar Typhimurium, SLDAPD, uses operator-repressor titration (ORT) technology, which requires only the short, nonexpressed lacO sequence for selection and maintenance. SLDAPD, recovered from the spleens and Peyer's patches of mice following oral inoculation, was shown to maintain a plasmid that, in contrast, was lost from parental strain SL3261. We also demonstrated successful application of this technology to vaccine development, since SLDAPD carrying a plasmid without an antibiotic resistance gene that expressed the Yersinia pestis F1 antigen was as efficacious in protecting vaccinated mice against plague as the parental SL3261 strain carrying an antibiotic-selected version of this plasmid. Protection of mice against plague by immunization with Salmonella expressing F1 has previously required two or more doses; here we demonstrated for the first time protective immunity after a single oral immunization. This technology can easily be used to convert any suitable attenuated strain to an antibiotic-free ORT strain for recombinant protein vaccine delivery in humans.

Efficacy of DNA vaccines expressing the type F botulinum toxin Hc fragment using different promoters.

DNA vaccines which expressed the Hc fragment of the Clostridium botulinum type F neurotoxin (BoNT/F Hc) fused to a signal peptide downstream of four different eukaryotic promoters were prepared. Subsequently, the immunogenicity of the DNA vaccines and protection afforded in mice against challenge with 10(4) MLD of type F botulinum toxin was evaluated. The DNA vaccine containing the human ubiquitin gene (UbC) promoter induced the highest BoNT/F Hc-specific antibody concentration following two intramuscular immunisations and afforded 90% protection against challenge. The results from this study indicate that the selection of promoter used in DNA vaccination studies may be of importance in designing optimised vaccines.

A Salmonella enterica serovar Typhi vaccine expressing Yersinia pestis F1 antigen on its surface provides protection against plague in mice.

A recombinant strain of attenuated Salmonella enterica serovar Typhi surface-expressing Yersinia pestis F1 antigen was generated by transforming strain BRD1116 (aroA aroC htrA) with plasmid pAH34L encoding the Y. pestis caf operon. BRD1116/pAH34L was stable in vitro and in vivo. An immunisation regimen of two intranasal doses of 1 x 10(8) cfu of BRD1116/pAH34L given intranasally to mice 7 days apart induced the strongest immune response compared to other regimens and protected 13 out of 20 mice from lethal challenge with Y. pestis. Intranasal immunisation of mice constitutes a model for oral immunisation with Salmonella vaccines in humans. Thus, the results demonstrate that attenuated strains of S. enterica serovar Typhi which express Y. pestis F1 antigen may be developed to provide an oral vaccine against plague suitable for use in humans.

Protection against plague afforded by immunisation with DNA vaccines optimised for expression of the Yersinia pestis V antigen.

DNA vaccine vectors were produced which were optimised for expression of the Yersinia pestis V antigen in the BALB/c mouse model. Six different eukaryotic promoters were compared, resulting in the selection of the CMV promoter with an additional translational enhancer downstream. Surprisingly, alteration of the codon usage of the lcrV gene encoding V antigen for expression in murine cells was not found to improve the antibody responses generated against V antigen. The DNA vaccine was subsequently evaluated in its delivery via intramuscular injection compared to gene-gun administration. Gene-gun delivery was found to induce significantly higher V antigen-specific antibody responses and also afforded the highest level of protection against Y. pestis challenge. In addition, the protection achieved could be increased by using a 'prime and boost' strategy, administering the DNA vaccine followed by recombinant V antigen. These results show promise for a DNA vaccine against plague.

DNA vaccines: improving expression of antigens.

DNA vaccination is a relatively recent development in vaccine methodology. It is now possible to undertake a rational step-by-step approach to DNA vaccine design. Strategies may include the incorporation of immunostimulatory sequences in the backbone of the plasmid, co-expression of stimulatory molecules, utilisation of localisation/secretory signals, and utilisation of the appropriate delivery system, for example. However, another important consideration is the utilisation of methods designed to optimise transgene expression. In this review we discuss the importance of regulatory elements, kozak sequences and codon optimisation in transgene expression.

Genotyping and phenotyping of beta2-toxigenic Clostridium perfringens fecal isolates associated with gastrointestinal diseases in piglets.

Although Clostridium perfringens is recognized as an important cause of clostridial enteric diseases, only limited knowledge exists concerning the association of particular C. perfringens toxinotypes (type A to E) with gastrointestinal (GI) diseases in domestic animals. Some C. perfringens isolates also produce the newly discovered beta2-toxin (CPB2). Recent epidemiological studies suggested that C. perfringens isolates carrying the gene encoding CPB2 (cpb2) are strongly associated with clostridial GI diseases in domestic animals, including necrotic enteritis in piglets and typhlocolitis in horses. These putative relationships, obtained by PCR genotyping, were tested in the present study by further genotyping and phenotyping of 29 cpb2-positive C. perfringens isolates from pigs with GI disease (pig GI disease isolates). PCR and restriction fragment length polymorphism analysis reconfirmed the presence of cpb2 gene sequences in all the disease isolates included in the study. Furthermore, genotyping by pulsed-field gel electrophoresis analyses showed that the pig GI disease isolates included in this study all carry a plasmid cpb2 gene, yet no clonal relationships were detected between the cpb2-positive pig GI disease isolates surveyed. Finally, CPB2-specific Western blotting demonstrated CPB2 expression by all of the cpb2-positive isolates surveyed. The CPB2 proteins made by five of these pig GI disease isolates were shown to have the same deduced amino acid sequences as the biologically active CPB2 protein made by the original type C isolate, CWC245. Collectively, our present results support a significant association between CPB2-positive C. perfringens isolates and diarrhea in piglets.

Salmonella enterica serovar typhimurium expressing a chromosomally integrated copy of the Bacillus anthracis protective antigen gene protects mice against an anthrax spore challenge.

Protective immunity against infection with Bacillus anthracis is almost entirely based on a response to the protective antigen (PA), the binding moiety for the two other toxin components. We cloned the PA gene into an auxotrophic mutant of Salmonella enterica serovar Typhimurium as a fusion with the signal sequence of the hemolysin (Hly) A gene of Escherichia coli to allow the export of PA via the Hly export system. To stabilize the export cassette, it was also integrated into the chromosome of the live Salmonella carrier. When S. enterica serovar Typhimurium with the chromosomally integrated PA gene was given intravenously to A/J mice, they developed high levels of antibody to PA. These mice were protected against intraperitoneal challenge with 100 or 1,000 50% lethal doses of B. anthracis strain STI. This work contributes to the development of a Salmonella-based orally delivered anthrax vaccine.

Oral immunisation with live aroA attenuated Salmonella enterica serovar Typhimurium expressing the Yersinia pestis V antigen protects mice against plague.

Bubonic and pneumonic plague are caused by the bacterium Yersinia pestis. The V antigen of Y. pestis is a protective antigen against plague. In this study, an aroA attenuated strain of Salmonella enterica serovar Typhimurium (SL3261) has been used to deliver the Y. pestis V antigen as a candidate oral plague vaccine. SL3261 was transformed with the expression plasmid pTrc-LcrV, containing the lcrV gene encoding V antigen. Immunoblot analysis showed V antigen expression in SL3261 in vitro and intragastric immunisation of mice with the recombinant Salmonella resulted in the induction of V antigen-specific serum antibody responses and afforded protection against Y. pestis challenge. However, the antibody responses induced by the recombinant Salmonella did not correlate with the protection afforded, indicating that immune responses other than antibody may play a role in the protection afforded against plague by this candidate vaccine.

Construction and evaluation of a eukaryotic expression plasmid for stable delivery using attenuated Salmonella.

An approach to enhancing the stability of eukaryotic expression plasmids for delivery using attenuated Salmonella has been evaluated. The expression apparatus and beta-galactosidase gene from the expression plasmid, pCMVbeta, was cloned into the low copy number plasmid pLG339. The resulting construct, pLGbetaGAL, was shown to have a lower copy number than pCMVbeta in Salmonella enterica var Typhimurium aroA strain SL7207. Furthermore, beta-galactosidase-specific antibody was induced in mice following intramuscular inoculation with pLGbetaGAL as naked DNA. Following oral administration of mice with SL7207/pCMVbeta, recombinants could not be detected in tissues 3 days after inoculation. In comparison, SL7207/pLGbetaGAL recombinant bacteria could be detected in the Peyer's patches and spleens indicating that the Salmonella strain was stable. However, both SL7207/pCMVbeta and SL7207/pLGbetaGAL failed to induce beta-galactosidase-specific IgG in vivo. The mechanism by which attenuated Salmonella are able to release heterologous DNA for antigen processing and presentation is not yet understood. These results suggest that the mechanism needs to be further elucidated in order to rationally improve the system.

Vaccination against type F botulinum toxin using attenuated Salmonella enterica var Typhimurium strains expressing the BoNT/F H(C) fragment.

The utility of the htrA, pagC and nirB promoters to direct the expression of the carboxy-terminal (H(C)) fragment of botulinum toxin F (FH(C)) in Salmonella enterica var Typhimurium has been evaluated. Only low levels of serum antibody were induced after immunisation, and some protection against botulinum toxin type F was demonstrated after oral immunisation of mice with two doses of any of these recombinant Salmonella. Immunisation with two doses of recombinant Salmonella expressing FH(C) from the htrA promoter gave the greatest protection, against up to 10,000 mouse lethal doses of botulinum toxin type F. These results demonstrate the feasibility of an orally delivered vaccine against botulinum toxin type F.

The use of live attenuated bacteria as a delivery system for heterologous antigens.

Live attenuated mutants of several pathogenic bacteria have been exploited as potential vaccine vectors for heterologous antigen delivery by the mucosal route. Such live vectors offer the advantage of potential delivery in a single oral, intranasal or inhalational dose, stimulating both systemic and mucosal immune responses. Over the years, a range of strategies have been developed to allow controlled and stable delivery of antigens and improved immunogenicity where required. Most of these approaches have been evaluated in Salmonella vaccine vectors and, as a result, several live attenuated recombinant Salmonella vaccines are now in human clinical trials. In this review, these strategies and their use in the development of a delivery system for the Yersinia pestis V antigen are described.