RESUMEN

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.

Asunto(s)

Antígenos Heterófilos/inmunología , Polisacáridos Bacterianos , Fiebre Tifoidea/prevención & control , Vacunas Tifoides-Paratifoides , Vacunas Atenuadas , Animales , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/inmunología , Vectores Genéticos , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/inmunología , Humanos , Salmonella typhi/genética , Salmonella typhi/inmunología

RESUMEN

Live, attenuated bacterial vaccines (LBV) are promising candidates for the induction of a broad-based immune response directed at recombinant heterologous antigens and the corresponding pathogen. LBVs allow vaccination through the mucosal surfaces and specific targeting of professional antigen-presenting cells located at the inductive sites of the immune system. A novel approach exploits attenuated intracellular bacteria as delivery vectors for eukaryotic antigen-expression plasmids (so-called DNA vaccines). Candidate carrier bacteria include attenuated strains of Gram-positive and Gram-negative bacteria. These bacteria have been shown to deliver DNA vaccines to human cells in vitro and have also proven their in vivo efficacy in several experimental animal models of infectious diseases and different cancers. The clinical assessment of the safety, immunogenicity and efficacy of these candidate strains will be the next challenging step towards live bacterial DNA vaccines.

Asunto(s)

Vacunas Bacterianas/genética , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Vacunas de ADN/genética , Animales , Vacunas Bacterianas/administración & dosificación , Enfermedades Transmisibles/tratamiento farmacológico , Enfermedades Transmisibles/genética , Técnicas de Transferencia de Gen/tendencias , Vectores Genéticos/administración & dosificación , Humanos , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Vacunas de ADN/administración & dosificación

RESUMEN

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.

Asunto(s)

Antígenos Bacterianos/genética , Escherichia coli/metabolismo , Vibrio cholerae/genética , Cromosomas Bacterianos , Ensayo de Inmunoadsorción Enzimática , Dosificación de Gen , Microscopía Electrónica , Operón , Plásmidos

RESUMEN

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.

Asunto(s)

Inmunidad Mucosa/inmunología , Salmonella/inmunología , Vacunación/métodos , Vacunas Atenuadas/inmunología , Animales , Antígenos/genética , Antígenos/inmunología , Antígenos/metabolismo , Vacunas Bacterianas/inmunología , Ensayos Clínicos como Asunto , Modelos Animales de Enfermedad , Vías de Administración de Medicamentos , Regulación Bacteriana de la Expresión Génica/genética , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Plásmidos/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Salmonella/genética , Salmonella/patogenicidad , Infecciones por Salmonella/etiología , Infecciones por Salmonella/inmunología , Salmonella enterica/inmunología , Salmonella enterica/patogenicidad , Salmonella typhi/genética , Salmonella typhi/inmunología , Salmonella typhi/patogenicidad , Salmonella typhimurium/genética , Salmonella typhimurium/inmunología , Transformación Bacteriana

RESUMEN

Live carrier-based bacterial vaccines represent a vaccine strategy that offers exceptional flexibility. Commensal or attenuated strains of pathogenic bacteria can be used as live carriers to present foreign antigens from unrelated pathogens to the immune system, with the aim of eliciting protective immune responses. As for oral immunisation, such an approach obviates the usual loss of antigen integrity observed during gastrointestinal passage and allows the delivery of a sufficient antigen dose to the mucosal immune system. Antibiotic and antibiotic-resistance genes have traditionally been used for the maintenance of recombinant plasmid vectors in bacteria used for biotechnological purposes. However, their continued use may appear undesirable in the field of live carrier-based vaccine development. This review focuses on strategies to omit antibiotic resistance determinants in live bacterial vaccines and discusses several balanced lethal-plasmid stabilisation systems with respect to maintenance of plasmid inheritance and antigenicity of plasmid-encoded antigen in vivo.

Asunto(s)

Vacunas Bacterianas/genética , Plásmidos/genética , Administración Oral , Animales , Antitoxinas/genética , Antitoxinas/inmunología , Toxinas Bacterianas/genética , Toxinas Bacterianas/inmunología , Vacunas Bacterianas/administración & dosificación , Farmacorresistencia Bacteriana/genética , Técnicas Genéticas , Humanos , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genética , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética

RESUMEN

This study examined the suitability of the hemolysin secretion system of Escherichia coli for expression and delivery of alpha-hemolysin (HlyA) by the S. typhi Ty21a strain, the only live oral Salmonella vaccine strain licensed for human use, under in vitro and in vivo conditions. For this purpose, two plasmid vectors encoding either the whole alpha-hemolysin of E. coli (pANN202-812/pMOhly2) or the hemolysin secretion signal (pMOhly1) were transferred into S. typhi Ty21a. S. typhi Ty21a carrying pANN202-812/pMOhly2 revealed efficient secretion of hemolysin in vitro. After formulation according to a process suitable for commercial production of Salmonella-based live bacterial vaccines, plasmids were shown to be stable in Ty21a and hemolysin secretion was demonstrated even after storage of the strains under real-time and stress conditions. After intranasal immunization of mice with S. typhi Ty21a/pANN202-812 plasmids are stable in vivo, and immunization induced a profound immune response against the heterologous HlyA antigen. Therefore, the combination of the hemolysin secretion system and S. typhi Ty21a could form the basis for a new generation of live bacterial vaccines.

Asunto(s)

Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/metabolismo , Proteínas de Escherichia coli/inmunología , Proteínas de Escherichia coli/metabolismo , Proteínas Hemolisinas/inmunología , Vacunas contra la Salmonella/administración & dosificación , Salmonella typhi/metabolismo , Animales , Antígenos Bacterianos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Vectores Genéticos , Proteínas Hemolisinas/genética , Inmunización , Ratones , Ratones Endogámicos C57BL , Plásmidos , Vacunas contra la Salmonella/genética , Salmonella typhi/genética

RESUMEN

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.

Asunto(s)

Bacterias/genética , Bacterias/inmunología , Técnicas de Transferencia de Gen , Vectores Genéticos/administración & dosificación , Plásmidos/genética , Vacunas de ADN/administración & dosificación , Animales , Vacunas Bacterianas/genética , Vacunas Bacterianas/inmunología , Vacunas contra el Cáncer/uso terapéutico , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Humanos , Técnicas In Vitro , Neoplasias/terapia , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas Atenuadas/uso terapéutico , Vacunas de ADN/genética , Vacunas de ADN/inmunología

RESUMEN

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.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Vacunas Bacterianas/inmunología , Listeriosis/prevención & control , Salmonella enteritidis/metabolismo , Administración Oral , Animales , Proteínas Bacterianas/inmunología , Epítopos de Linfocito B/genética , Epítopos de Linfocito B/metabolismo , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/metabolismo , Escherichia coli , Proteínas de Escherichia coli , Técnica del Anticuerpo Fluorescente , Listeria monocytogenes/genética , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Proteínas de Transporte de Membrana , Ratones , Ratones Endogámicos BALB C , Plásmidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salmonella enteritidis/genética , Vacunas Atenuadas/inmunología

RESUMEN

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.

Asunto(s)

Antígenos Bacterianos/biosíntesis , Vectores Genéticos , Vacunas de ADN/biosíntesis , Animales , Antígenos Bacterianos/genética , Escherichia coli/genética , Escherichia coli/inmunología , Células Eucariotas/inmunología , Bacterias Gramnegativas/inmunología , Proteínas Hemolisinas/biosíntesis , Proteínas Hemolisinas/genética , Humanos , Listeria monocytogenes/inmunología , Vacunas Atenuadas , Vacunas de ADN/genética , Vacunas Sintéticas