Simone Spreng

Recombinant attenuated bacteria for the delivery of subunit vaccines

Using attenuated intracellular bacteria as carriers, we have developed two different approaches for the delivery of subunit vaccines encoding heterologous antigens. The first system is based on the direct secretion of the heterologous antigens in Gram-negative bacteria via the hemolysin secretion system of Escherichia coli into either phagosome or cytosol of infected cells. The second approach is based on the transport of eukaryotic antigen expression vectors by intracellular bacteria like Listeria and Salmonella into the host cell and here, preferably, into the cytosolic compartment. After release of the plasmid DNA from the bacteria, the plasmid-encoded antigens can be expressed directly by the host cell. Finally, we combined both types of subunit vaccines in one live vector - we equipped Salmonella strains with a phagosomal escape function by utilization of the hemolysin secretion system and used this recombinant vaccine strain for the delivery of a eukaryotic antigen expression vector into the cytosol of macrophages.

Use of attenuated bacteria as delivery vectors for DNA vaccines

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.

Delivery of protein antigens and DNA by virulence-attenuated strains of Salmonella typhimurium and Listeria monocytogenes.

Two different plasmid-vector systems were developed which allow the efficient production and presentation of protein antigens in antigen-presenting cells (APC) by means of virulence-attenuated bacteria. The first antigen-delivery system is based on the secretion machinery of the Escherichia coli hemolysin (HlyA-type I secretion system), which transports proteins, possessing the specific HlyA secretion signal (HlyA(s)) at the C-terminus, across both membranes of gram-negative bacteria. This system functions in all gram-negative bacteria that possess the TolC-analogous protein in the outer membrane. This outer membrane protein is necessary for the stable anchoring of the type I secretion apparatus in the cell envelope. Suitable HlyA(s)-fused antigens are secreted with high efficiency by E. coli and by virulence-attenuated strains of Salmonella, Shigella, Vibrio cholerae and Yersinia enterocolitica. The other vector system expresses the heterologous antigen under the control of an eukaryotic promoter in a similar fashion as in plasmids commonly used for vaccination with naked DNA. This plasmid DNA is introduced into APCs with the help of virulence-attenuated self-destructing Listeria monocytogenes mutants. After synthesis of the heterologous protein, epitopes of the antigen are presented by the APC together with MHC class I molecules. This system functions in macrophages and dendritic cells in vitro and can also be used in a modified form in animal models.

Cancer immunotherapy based on recombinant Salmonella enterica serovar Typhimurium aroA strains secreting prostate-specific antigen and cholera toxin subunit B

Prostate cancer is the most common malignant tumor in men and is normally associated with increased serum levels of prostate-specific antigen (PSA). Therefore, PSA is one potential target for a prostate cancer vaccine. In this study we analyzed the functionality of new bacterial PSA vaccines, expressed and secreted via the hemolysin (HlyA) secretion system of Escherichia coli, the prototype of Type I secretion systems (T1SS) using an attenuated Salmonella enterica serovar Typhimurium aroA strain as carrier. The data demonstrate that a bacterial live vaccine encompassing T1SS in combination with cholera toxin subunit B can be successfully used for delivery of PSA to induce cytotoxic CD8+ T-cell responses resulting in an efficient prevention of tumor growth in mice.

Gram-positive and Gram-negative bacteria as carrier systems for DNA vaccines

Vaccination by intradermal or intramuscular injection of eukaryotic antigen expression vectors (so-called DNA vaccines) elicits strong cellular and humoral immune responses. A novel approach employs attenuated mutant strains of Gram-positive and Gram-negative intracellular bacteria as carriers for the delivery of DNA vaccines. This strategy allows the administration of the DNA vaccines via mucosal surfaces and a direct delivery of the plasmid DNA to professional antigen presenting cells (APC), such as macrophages and dendritic cells (DC). In this work, we have found that several Gram-negative bacteria are capable of delivering plasmid vectors to human DC. In addition, we tested the suitability of the Gram-positive bacterium Listeria monocytogenes as a vaccine carrier for the immunization of fish.

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.

Salmonella vaccines secreting measles virus epitopes induce protective immune responses against measles virus encephalitis.

In the present study we describe a live vaccine against measles virus (MV) infection on the basis of attenuated Salmonella typhimurium aroA secreting MV antigens via the Escherichia coli alpha-hemolysin secretion system. Two well-characterized MV epitopes, a B-cell epitope of the MV fusion protein (amino acids 404-414) and a T-cell epitope of the MV nucleocapsid protein (amino acids 79-99) were fused as single or repeating units to the C-terminal secretion signal of the E. coli hemolysin and expressed in secreted form by the attenuated S. typhimurium aroA SL7207. Immunization of MV-susceptible C3H mice revealed that S. typhimurium SL7207 secreting these antigens provoked a humoral and a cellular MV-specific immune response, respectively. Mice vaccinated orally with a combination of both recombinant S. typhimurium strains showed partial protection against a lethal MV encephalitis after intracerebral challenge with a rodent-adapted, neurotropic MV strain.

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.

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.