Improvement of the live vaccine strain Salmonella enterica serovar Typhi Ty21a for antigen delivery via the hemolysin secretion system of Escherichia coli.

The attenuated Salmonella enterica serovar Typhi strain Ty21a (Ty21a) is the only attenuated live oral vaccine against typhoid fever. Ty21a is also an attractive carrier for the delivery of heterologous antigens. We have used Ty21a for antigen delivery via the hemolysin (HlyA) secretion system of Escherichia coli, the prototype of the type I secretion system (T1SS). In this study, we identified by genetic complementation that the specific mutation of rpoS correlated with the hemolysin production of strain Ty21a. We furthermore showed that complementation with a plasmid encoding rfaH, which is described to be a downstream target of rpoS, led to increased expression and secretion of hemolysin. Finally, we demonstrated a significant enhancement of antibody responses against the heterologous HlyA antigen of Ty21a after immunization of mice with rfaH complemented S. typhi strain secreting HlyA compared with the same strain without rfaH plasmid.

Genetic stability of vaccine strain Salmonella Typhi Ty21a over 25 years.

The attenuated live bacterial vaccine strain Salmonella enterica Serovar Typhi Ty21a is the main constituent of Vivotif, the only licensed oral vaccine against typhoid fever. The strain was developed in the 1970s by chemical mutagenesis. In the course of this mutagenesis, a number of mutations were introduced into the vaccine strain. Characterisation of the vaccine strain during development as well as release of master- and working seed lots (MSL and WSL) and commercial batches is based on phenotypic assays assessing microbiological and biochemical characteristics of Ty21a. In the current study, we have analysed by DNA sequencing the specific mutations originally correlated with the attenuation of strain Ty21a. These data demonstrate the stability of these mutations for MSLs and WSLs of Ty21a produced between 1980 and 2005. Finally, we have confirmed the correlation of these genetic mutations with the expected phenotypic attenuations for the seed lots used in vaccine manufacture over 25 years.

Vivotif--a 'magic shield' for protection against typhoid fever and delivery of heterologous antigens.

The attenuated Salmonella typhi strain Ty21a is the main constituent of Vivotif, the only attenuated live oral vaccine against typhoid fever. In comparison with antibiotics, the 'magic bullets' which Paul Ehrlich was striving for to treat infectious diseases, this vaccine should be viewed as a 'magic shield', because rather than treating typhoid fever after the infection has started, immunisation with this vaccine strain prevents infection and disease by the induction of specific immune responses. Ty21a is also an attractive carrier for the delivery of heterologous antigens. Recently, we successfully used Ty21a for antigen delivery via the haemolysin secretion system of Escherichia coli, which allows efficient protein secretion from the carrier bacteria.

The E. coli alpha-hemolysin secretion system and its use in vaccine development.

Many Gram-negative bacteria use a type I secretion system to translocate proteins, including pore-forming toxins, proteases, lipases and S-layer proteins, across both the inner and outer membranes into the extracellular surroundings. The Escherichia coli alpha-hemolysin (HlyA) secretion system is the prototypical and best characterized type I secretion system. The structure and function of the components of the HlyA secretion apparatus, HlyB, HlyD and TolC, have been studied in great detail. The functional characteristics of this secretion system enable it to be used in a variety of different applications, including the presentation of heterologous antigens in live-attenuated bacterial vaccines. Such vaccines can be an effective delivery system for heterologous antigens, and the use of a type I secretion system allows the antigens to be actively exported from the cytoplasm of the bacterial carrier rather than only becoming accessible to the host immune system after bacterial disintegration.

Haemolysin A and listeriolysin--two vaccine delivery tools for the induction of cell-mediated immunity.

Haemolysin A of Escherichia coli and listeriolysin of Listeria monocytogenes represent important bacterial virulence factors. While such cytolysins are usually the reason for morbidity and even mortality, vaccine researchers have turned haemolysin A and listeriolysin into tools for vaccine delivery. Both cytolysins have found widespread application in vaccine research and are highly suitable for the elicitation of cell-mediated immunity. In this paper, we will review vaccine delivery mediated by the haemolysin A secretion system and listeriolysin and will highlight their use in vaccination approaches against protozoan parasites.

DNA microarrays in vaccine research.

DNA microarrays represent the technology platform for a wide variety of analytical methods built around the detection of sequence-specific nucleic acid hybridization. They allow the analysis of complex biological systems on the basis of the genome and transcriptome with high simplicity, efficiency, sensitivity and specificity. This positions DNA microarrays as ideal tools for novel approaches in biomedical research and explains the tremendous pace at which they are being applied to an enormous variety of systems and projects. Recently, vaccine researchers began to use DNA microarrays for the identification of vaccine candidates against bacterial, parasitic and viral pathogens, as well as tumors. Here, DNA microarrays as a technology platform in these areas of vaccine research are reviewed and their limitations, as well as their potential for future applications in vaccinology, critically evaluated.

Live attenuated bacteria as vectors to deliver plasmid DNA vaccines.

Live attenuated bacterial vaccines allow vaccination via the mucosal surfaces and specific targeting to professional antigen presenting cells located at the inductive sites of the immune system. A novel approach exploits attenuated intracellular bacteria as a delivery system for eukaryotic antigen expression vectors (so-called DNA vaccines). Candidate carrier bacteria include attenuated strains of Salmonella, Shigella and Listeria spp, which have been shown, in vitro, to deliver DNA vaccines to human cells. Bacterial DNA vaccine delivery has also demonstrated in vivo efficacy in several experimental animal models of infectious diseases and tumors. The next step should be the clinical assessment of the safety, immunogenicity and efficacy of DNA vaccine delivery by live bacterial vaccines.

Cultivation of Mycobacterium bovis BCG in bioreactors.

The Mycobacterium bovis BCG vaccine for commercial use is classically produced as surface pellicles by culture on synthetic medium. Under these conditions, reproducibility of the cultures and quality assessment are hampered by slow growth of the bacilli, the formation of bacterial aggregates and a high proportion of dead bacilli after processing and final formulation of the vaccine. Here, we established dispersed cultures of M. bovis BCG in synthetic media in small-scale bioreactors. These cultures allow recording and adjusting of culture parameters and give rise to single bacilli with a high degree of live bacteria. In the murine model, bioreactor-grown M. bovis BCG exhibited slightly stronger replication and persistence than the vaccine produced under the classical conditions. The protective efficacy against challenge with M. tuberculosis was identical for both vaccine preparations.

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.

Genomic changes during chronic Helicobacter pylori infection.

The gastric pathogen Helicobacter pylori shows tremendous genetic variability within human populations, both in gene content and at the sequence level. We investigated how this variability arises by comparing the genome content of 21 closely related pairs of isolates taken from the same patient at different time points. The comparisons were performed by hybridization with whole-genome DNA microarrays. All loci where microarrays indicated a genomic change were sequenced to confirm the events. The number of genomic changes was compared to the number of homologous replacement events without loss or gain of genes that we had previously determined by multilocus sequence analysis and mathematical modeling based on the sequence data. Our analysis showed that the great majority of genetic changes were due to homologous recombination, with 1/650 events leading to a net gain or loss of genes. These results suggest that adaptation of H. pylori to the host individual may principally occur through sequence changes rather than loss or gain of genes.

Vaccines against typhoid fever.

Because of high infectivity and significant disease burden, typhoid fever constitutes a major global health problem. Implementation of adequate food handling practices and establishment of safe water supplies are the cornerstone for the development of an effective prevention program. However, vaccination against typhoid fever remains an essential tool for the effective management of this disease. Currently, there are two well tolerated and effective licensed vaccines. One is based on defined subunit virulence (Vi) polysaccharide antigen and can be administered either intramuscularly or subcutaneously and the other is based on the use of live attenuated bacteria for oral administration. The advantages and disadvantages of the various approaches taken in the development of a vaccine against typhoid fever are discussed, along with the potential for future vaccine candidates.

Expression of enterotoxigenic Escherichia coli colonization factors in Vibrio cholerae.

As a first step towards a vaccine against diarrhoeal disease caused by enterotoxigenic Escherichia coli (ETEC), we have studied the expression of several ETEC antigens in the live attenuated Vibrio cholerae vaccine strain CVD 103-HgR. Colonization factors (CF) CFA/I, CS3, and CS6 were expressed at the surface of V. cholerae CVD 103-HgR. Both CFA/I and CS3 required the co-expression of a positive regulator for expression, while CS6 was expressed without regulation. Up-regulation of CF expression in V. cholerae was very efficient, so that high amounts of CFA/I and CS3 similar to those in wild-type ETEC were synthesized from chromosomally integrated CF and positive regulator loci. Increasing either the operon and/or the positive regulator gene dosage resulted in only a small increase in CFA/I and CS3 expression. In contrast, the level of expression of the non-regulated CS6 fimbriae appeared to be more dependent on gene dosage. While CF expression in wild-type ETEC is known to be tightly thermoregulated and medium dependent, it seems to be less stringent in V. cholerae. Finally, co-expression of two or three CFs in the same strain was efficient even under the control of one single regulator gene.

Novel vaccination strategies based on recombinant Mycobacterium bovis BCG.

In this manuscript, we will review the utilization of Mycobacterium bovis Bacille Calmette-Guerin (BCG) as a vaccine against tuberculosis (TB) and as a carrier system for heterologous antigens. BCG is one of the most widely used vaccines. Novel techniques in genome manipulation allow the construction of virulence-attenuated recombinant (r)-BCG strains that can be employed as homologous vaccines, or as heterologous antigen delivery systems, for priming pathogen-specific immunity against infectious diseases, including TB. Several approaches are available for heterologous antigen expression and compartmentalization in BCG and recent findings show the potential to modulate and direct the immune responses induced by r-BCG strains as desired. Recent achievements in complete genome analysis of various target pathogens, combined with a better understanding of protective pathogen-specific immune responses, form the basis for the rational design of a new generation of recombinant mycobacterial vaccines against a multitude of infectious diseases.

Vaccines against Infections Caused by Salmonella, Shigella, and Pathogenic Escherichia coli.

Infectious diseases represent one of the most common causes of death worldwide, with the enteropathogenic bacteria Salmonella and Shigella and pathogenic Escherichia coli being among the most detrimental. Currently, vaccination represents the preferred method of preventing such infections. For stimulating the adaptive immune response, immunizations are frequently based on formulations which include inactivated whole-cell vaccines, live attenuated vaccines, or subunit vaccines. These can be administered via a parenteral or mucosal route, the latter having the advantage that it most closely mimics the actual course of infection. In addition to the type of vaccine and method of application, important consideration needs to be paid to safety, efficacy, and cost, which are often major bottlenecks in the successful implementation of vaccines. In this chapter we take a limited look at the history surrounding vaccinations involving Salmonella, Shigella, and pathogenic E. coli. Salmonella infections, which can lead to typhoid fever, are becoming increasing difficult to treat with antibiotics due to multi-drug-resistant strains. At present, the parenteral Vi-based subunit vaccines and the live attenuated oral vaccine Ty21a have proven to be the vaccines of choice, with high levels of protective efficacy and limited side effects. Shigella infections are responsible for the diarrheal disease shigellosis. Various live and nonliving mucosal and parenteral vaccines have been tested, with the most promising candidates evolving around those that stimulate the production of O-antigen-specific antibodies. Pathogenic Escherichia coli infections can lead to severe diseases due to the bacterium's production of several specific toxins. Vaccines against this bacterium target its toxins, as well as surface-exposed antigens, all of which have been found to be effective as immunogens.

Biosafety aspects of the recombinant live oral Vibrio cholerae vaccine strain CVD 103-HgR.

The development of live attenuated vaccines, allowing for the safe and effective immunisation at mucosal surfaces, is a strategy of great interest for vaccinologists. The main advantage of this approach over conventional parenteral vaccines is the induction of strong mucosal immune responses, allowing targeting of the pathogen at the initial point of contact with the host. Further advantages include the ease of administration, high acceptance by vaccines, and relatively low production costs. Finally, well-characterised, safe and immunogenic vaccine strains are well suited as vectors for the mucosal delivery of foreign vaccine antigens and of DNA vaccines. However, such vaccines, when based on or containing genetically modified organisms (GMOs), are facing new and specific regulatory hurdles, particularly regarding the potential risks for humans and the environment. In this contribution we address selected aspects of the risk assessment of live attenuated bacterial vaccines covered in the course of the registration of vaccine strain CVD 103-HgR as a recombinant live oral vaccine against cholera.

Mycobacterium bovis BCG-based vaccines against tuberculosis: novel developments.

Mycobacterium bovis Bacille Calmette-Guérin (BCG) is one of the most widely used vaccines. Modern techniques in genome manipulation allow the construction of recombinant (r)-BCG strains that can be employed as highly immunogenic vaccines against tuberculosis (TB) with an enhanced safety profile. In addition, the development of novel procedures to cultivate BCG will allow the large-scale production of future BCG-based vaccines.

Protection against murine listeriosis by oral vaccination with recombinant Salmonella expressing protective listerial epitopes within a surface-exposed loop of the TolC-protein.

Based on the topology of the outer membrane protein TolC of Escherichia coli, a new plasmid-encoded system was created which allows the expression of antigenic peptides within permissive, surface-exposed domains of TolC. To assess the capacity of this novel antigen display system, a protective CD4 T-cell epitope of the p60 protein of Listeria monocytogenes was inserted within an extracellular loop of the TolC-protein and expressed in surface-exposed form by attenuated Salmonella enteritidis. Mice immunized orally with this recombinant S. enteritidis live vaccine strain were protected against a lethal challenge with wildtype L. monocytogenes.

Experience with registered mucosal vaccines.

Most pathogens gain access to their host through mucosal surfaces. It is therefore desirable to develop vaccination strategies that lead to mucosal immune responses. Ideally, a vaccine should be administered mucosally in order to elicit mucosal protection. Several attenuated live viral and bacterial pathogens are registered as oral vaccines for human use, including the oral polio vaccine (Sabin) as well as attenuated strains of Salmonella typhi and Vibrio cholerae. These attenuated bacterial live vaccines-S. typhi Ty21a as well as V. cholerae CVD 103-HgR-are employed as vaccines against typhoid and cholera, respectively. In this manuscript, we review the immune responses that are induced by these vaccines, with a focus on mucosal immunity.

Delivery of protein antigens and DNA by attenuated intracellular bacteria.

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 (type I) 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 and dendritic cells in vitro and can be also used in animal models.

Transcriptome-based antigen identification for Neisseria meningitidis.

The identification of suitable antigens is crucial to successful vaccine development based on subunit approaches. While many methods exist for the identification of vaccine candidates which are surface-exposed or secreted, immunogenic and conserved, contain B and T cell epitopes, most of these have a major drawback: they do not yield any information on whether the antigen is indeed expressed by the pathogen during infection. However, DNA microarrays offer a novel tool for the investigation of the transcriptional activity of all genes of a pathogenic microorganism under in vivo conditions. Employing whole genome DNA microarrays, we have analyzed the transcriptome of Neisseria meningitidis serogroup B bacteria during different stages of infection, i.e. exposure to human serum and the interaction with human epithelial and endothelial cells. Combined with data derived from genome-based approaches (such as reverse vaccinology) and immunogenicity studies, this novel transcriptome-based antigen identification should reveal ideal vaccine candidates against serogroup B meningococcal infection.