Transfer of eukaryotic expression plasmids to mammalian hosts by attenuated Salmonella spp.

Int. J. Med. Microbiol. 293, 95 ± 106 (2003) ¹ Urban & Fischer Verlag http: // www.urbanfischer.de/journals/ijmm

Transfer of eukaryotic expression plasmids to mammalian hosts by attenuated Salmonella spp. Siegfried Weiss Molecular Immunology, GBF, German Research Centre for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany

Abstract Transkingdom transfer of DNA from bacteria to other organisms, well established for bacteria, yeast and plants, was recently also extended to mammalian host cells. Attenuated intracellular bacteria or non-pathogenic bacteria equipped with adhesion and invasion properties have been demonstrated to transfer eukaryotic expression plasmids in vitro and in vivo. Here the mucosal application of attenuated Salmonella enterica spp. as DNA carrier for the induction of immune responses towards protein antigens encoded by expression plasmids, their use to complement genetic defects or deliver immunotherapeutic proteins is reviewed. Plasmid transfer has been reported for Salmonella typhimurium, S. typhi and S. choleraesuis so far but clearly other Salmonella strains should be able to transfer expression plasmids as well. Transfer of DNA is effected most likely by bacterial death within the host cell resulting from metabolic attenuation. Since these bacteria remain in the phagocytic vacuole it is unclear how the DNA from such dying bacteria is delivered to the nucleus of infected cells. Nevertheless, the efficiency that has been observed was astonishingly high, reaching close to 100% under certain conditions. Gene transfer in vivo was mainly directed towards vaccination strategies either as vaccination against infectious microorganisms or model tumors. Interestingly, in some cases tolerance against autologous antigens could be broken. In general, this type of immunization was more efficacious than either direct application of antigen, vaccination with naked DNA or using the same bacterium as a heterologous carrier expressing the antigen via a prokaryotic promoter. The ease of generating such vehicles for gene transfer combined with technology validated for mass vaccination programs and the efficacy of induction of protective immune responses makes Salmonella as carrier for mucosal DNA vaccination a highly attractive area for further research and development. Key words: Salmonella ± DNA transfer ± vaccination

Introduction Direct transfer of eukaryotic expression plasmids form bacteria to eukaryotic host cells has been already achieved several years ago by the fusion between mammalian cells and protoplasts of plas-

mid-carrying Escherichia coli (Schaffner, 1980). In the meantime it is firmly established that kingdom boundaries can be traversed in many directions by genetic information. Thus, plasmids were found to be transferred between Gram-negative and Gram-

Corresponding author: S. Weiss, Molecular Immunology, GBF, German Research Centre for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany. Phone: ‡ 49 5 31 61 81 2 30, Fax: ‡ 49 5 31 61 81 4 44, E-mail: [email protected]

1438-4221/03/293/01-095 $ 15.00/0

96

S. Weiss

positive bacteria as well as between bacteria and yeast (Heinemann and Sprague, 1989; Trieu-Cuot et al., 1993; Charpentier et al., 1999). In addition, bacteria-mediated DNA transfer to plants is well known and in use as standard methodology for the generation of recombinant plants which has been exploited by agricultural industry for almost 20 years by now (Lessl and Lanka, 1994). Expression plasmid transfer from viable bacteria to mammalian host cells has been discovered by four groups independently (Sizemore et al., 1995; Courvalin et al., 1995; Powell et al., 1996; Darji et al., 1997). Attenuated Shigella flexneri, Salmonella typhimurium or E. coli that had been rendered invasive by the virulence plasmid of S. flexneri were shown to be able to transfer expression plasmids after invasion of host cells and intracellular death due to the metabolic attenuation. Mucosal application either nasally or orally of such recombinant Shigella or Salmonella induced immune responses against the antigen that was encoded by the expression plasmids. In the meantime the list of bacteria that was shown to be able to transfer expression plasmids to mammalian host cells in vitro and in vivo has been more then doubled. Hence, such ability has been documented for S. typhi, S. choleraesuis, Listeria monocytogenes, Yersinia pseudotuberculosis, and Y. enterocolitica (Fennelly et al., 1999; Shiau et al., 2001; Dietrich et al., 1998, 2001; Hense et al., 2001; Al-Mariri et al., 2002). In general, one could assume that all bacteria that are able to enter the cytosol of the host cell, like S. flexneri or L. monocytogenes and lyse within this cellular compartment, should be able to transfer DNA. In addition, even many of the bacteria that remain in the phagocytic vacuole like S. typhimurium may also be able to do so. Thus, recombinant laboratory strains of E. coli that had been engineered to be invasive but unable of phagosomal escape, could deliver their plasmid load to the nucleus of the infected mammalian cell nevertheless (Grillot-Courvalin et al., 1998). Interestingly, Agrobacterium tumefaciens, a bacterium that is known to transfer DNA into plant cells, has recently also been shown to introduce transgenes into mammalian cells (Kunik et al., 2001). This bacterium attaches to the target cell and transfers DNA employing a conjugational apparatus without invasion. While in the latter case a special mechanism is responsible for DNA transfer, transfer of expression plasmids by the other types of bacteria might be rather unspecific. An obvious way of bacteriamediated transfection can be envisioned for bacteria that escape into the cytosol of the target cell. Upon

death and lysis of the intracytosolic bacteria which might be achieved by an auxotrophy for essential components that the host cells cannot supply (Sizemore et al., 1995; Courvalin et al., 1995; Powell et al., 1996), by the inducible expression of an autolysin (Dietrich et al., 1998) or simply by administration of appropriate antibiotics (Dietrich et al., 1998; Hense et al., 2001), the expression plasmids are liberated within this compartment and reach the nucleus, possibly by chance. During these processes the liberated plasmids could be stabilized by bacterial proteins that might still be associated with the plasmid DNA. This overall idea was strongly supported by the finding that plasmid DNA could be demonstrated in the culture supernatants of E. coli in which lysis had been induced by activating lytic components of bacteriophage l (Jain and Mekalanos, 2000). More astonishing is the ability of transfer of plasmid DNA to host cells by bacteria that remain in the phagocytic vacuole. It is unclear how the transfer is achieved but the efficiency can be extremely high reaching a transfection rate of close to 100% under certain circumstances in vitro (Montosi et al., 2000). It could be possible that the bacteria encode a machinery to transfer macromolecules into target cells. For example, the type III secretion mechanism found in several Gram-negative bacterial species is able to inject bacterial proteins into the cytosol of the host cell. A similar mechanism might be imagined for the crossing of the phagosomal membrane by plasmid DNA. That laboratory strains of E. coli (Grillot-Courvalin et al., 1998) are also capable of transferring expression plasmids does not necessarily argue against such a possibility. However, a certain extent of cell specificity has been observed for the different bacterial species in their ability to transfer DNA from the phagocytic vacuole. While E. coli was able to act as DNA carrier in established cell lines of epithelial origin (Grillot-Courvalin et al., 1998), S. typhimurium was found to exclusively transfer DNA into primary murine and human macrophages and human dendritic cells in vitro (Darji et al., 1997; Montosi et al., 2000; Dietrich et al., 2001). Transfer into established cell lines independent of their tissue origin was extremely inefficient (Darji et al., 1997; Grillot-Courvalin et al., 2002). In addition, the transfer of DNA by bacterial ghosts has been demonstrated (Lubitz, 2001), rendering an active mechanism encoded by the microorganisms highly improbable. Thus, it is more likely that particular host cell-specific pathways exist that are exploited by the different bacterial species. The potential of transfer of macromolecules from the vacuole of dendritic cells as well

DNA transfer by Salmonella

as of activated monocytes into the cytosol has recently been demonstrated (Norbury et al., 1995; Rodriguez et al., 1999). In addition, an unexpected connection between the endoplasmic reticulum and the phagosome as well as the plasma membrane has be described (Gagnon et al., 2002). Unknown and cell-specific transfer routes from vesicular compartments to the cytosol might exist and be employed by such bacteria. So far Salmonella have been used for the mucosal delivery of DNA vaccines, for the transfer of therapeutic molecules and for the complementation of monogenic defects. While the latter was restricted to particular situations only, the two other types of usage have successfully and extensively been employed in vivo and have demonstrated the general versatility of this system.

Salmonella-mediated mucosal DNA vaccination Prophylactic genetic vaccination against pathogens using attenuated Salmonella Most of the data on Salmonella-mediated mucosal DNA vaccination has been obtained using attenuated S. typhimurium as transfer system. Mice are a natural host of these bacteria, thus, experiments can be performed under physiological conditions. In addition, S. typhimurium is also applicable to humans as vaccine carrier and several strains have proved to be safe when administered (Mastroeni et al., 2000). In addition, S. typhi and S. choleraesuis have been proven by now to be able to act as carrier for mucosal DNA vaccines as well. Mainly commercially available vectors have been employed as eukaryotic expression plasmids. Such plasmids are based on the origin of replication (ori) of the pUC vector series that mediates the replication of the plasmids at a high copy number in the bacterial carrier. They usually carry an ampicillin resistance marker to select and stabilize the plasmid in culture. Originally, listeriolysin O and ActA two virulence factors of L. monocytogenes and b-galactosidase (bgal) of E. coli were successfully tested (Darji et al., 1997). Cytotoxic and helper T cells (mainly Th1cells) as well as specific antibodies could be detected against these antigens even after a single oral dose of the recombinant Salmonella. This type of administration was far superior to oral application of the same number of Salmonella that expressed high amounts of antigen as heterologous protein driven by a prokaryotic promoter. In addition, T cell memory could only be induced under

97

these circumstances by the oral application of recombinant Salmonella that carried the eukaryotic expression plasmid and not by the Salmonella expressing the antigen themselves (Darji et al., 2000). Immunization with Salmonella carrying an expression plasmid encoding listeriolysin, elicited a protective response against a lethal challenge with L. monocytogenes. These findings were obtained in BALB/c mice which are highly susceptible to Salmonella infection. Extension to strains of a more resistant phenotype and to outbred mice revealed that cytotoxic and helper T cell responses could be induced in all of these strains after a single application only. However, several administrations of the recombinant Salmonella were required to elicit specific antibody responses to the antigens studied ((Darji et al., 2000) and unpublished). This indicates that with the present carrier and expression plasmid combination mainly T cell responses are elicited while further improvements are required to generate strong systemic as well as local antibody responses. Nasal and oral application of Salmonella-based DNA vaccines encoding b-gal or a fusion antigen of the outer membrane protein and the fimbriae derived from Pseudomonas aeruginosa have been compared. Several nasal administrations were necessary to observe a T cell response in spleen compared to a single dose for oral administration ((Darji et al., 2000) and unpublished). Specific IgG could be observed in gut, saliva and serum after oral administration and in lung, saliva and serum after nasal administration. However, hardly any antigenspecific IgA could be detected ((Darji et al., 2000) and unpublished). Although many transgene-expressing cells were demonstrated in the Peyer's patches (PP) after oral Salmonella-mediated DNA transfer (Urashima et al., 2000), such cells might not support efficient antibody production and/or the switch of B cells to IgA. Mucosal T cell responses were not determined during these experiments. In the meantime several studies have extended the original findings. Partial protective responses were obtained against Chlamydia trachomatis using an expression plasmid encoding the major outer membrane protein as antigen (Brunham and Zhang, 1999). Interestingly, this protection was observed in the lung although the vaccine was administered orally. Similarly, Salmonella-mediated oral DNA vaccination using the glycoprotein D of Herpes simplex virus-2 as antigen resulted in induction of cytotoxic and helper T cells (Flo¬ et al., 2001). In addition, such mice were protected against a lethal intravaginal challenge with the virus and local IFN-g production could be observed in the mucosal tissue of this organ after the challenge. Such findings are in

98

S. Weiss

agreement with the recent observation that T cells disseminate into the tissue after activation in inductive lymphoid organs (Reinhardt et al., 2001) This, might explain the discrepancy with our findings on the lack of local antibody production (Darji et al., 2000). T cells and antibody responses could also be observed using the surface protein of hepatitis B virus as antigen (Woo et al., 2001). In this case, Salmonella-based oral DNA vaccination resulted in similar cytotoxic T cell responses as intramuscular application of the naked expression plasmid or the recombinant subunit vaccine. Antibody responses however were comparably low after oral administration of the recombinant bacteria. In vitro transfection of macrophages using the recombinant Salmonella allowed the quantitation of antigen expression. Obviously, only low protein expression is obtained under these circumstances in macrophages when compared with conventional transfectants. Nevertheless, the direct expression of the antigen in inductive cells of the immune system might more than compensate for such low expression levels of antigen. On the other hand, the low level of antigen expression might explain why antibody production is generally poor in this system and some mouse strains require more than one application for a significant antibody response to take place (Darji et al., 2000). When macrophages transfected via Salmonella were administered intraperitoneally or intramuscularly into mice, an immune response of the same quality was elicited as was observed with the normal oral application of the recombinant Salmonella (Zheng et al., 2001). Intriguing improvements on efficacy of Salmonella-mediated DNA vaccination were observed in HIV-specific responses. Administering recombinant Salmonella carrying a gp120-encoding plasmid required several intragastric administrations to obtain a significant CD8 T cell response. However, using an expression cassette for gp120 of HIV that was codon optimized for human vaccination already a single application sufficed (Shata et al., 2001). Strong induction of IFN-g production by CD8 Tcells could be demonstrated when cells were isolated from spleen as well as from PP. In contrast, such activity could only be revealed in spleen when the naked expression plasmid was applied intramuscularly. This suggests a local induction of the cytotoxic response. The restriction of DNA transfer mediated by S. typhimurium to primary macrophages and dendritic cells could limit the applicability of this system. As one possibility that was pursued to overcome such limitations (i.e. to allow plasmid transfer also into

established cell lines and possibly also to improve the performance of this carrier in vivo), Salmonella were engineered to secrete listeriolysin O the pore-forming toxin of L. monocytogenes (Gentschev et al., 1995). These bacteria are able to escape from the phagocytic vacuole into the cytosol of the host cell. Co-infection of primary macrophages with Salmonella secreting listeriolysin and Salmonella carrying an ovalbumin-encoding expression plasmid considerably enhanced antigen expression in these host cells (Catic et al., 1999). Similar enhancements of reporter gene expression were observed in vivo in the peritoneum of mice three days after oral administration of Salmonella secreting listeriolysin and carrying a GFP-encoding expression plasmid at the same time (Dietrich et al., 2000). In the meantime also attenuated strains of S. typhi have been employed as carrier for mucosal DNA vaccination. S. typhi is not virulent in mice, therefore, studies using these bacteria as DNA carrier are limited to nasal or intraperitoneal administration in such animals. Approved vaccine strains already exist, thus, clinical trials would be extremely facilitated using such strains. However, a proof of principle using these bacteria as carriers for mucosal DNA vaccination was required. The strain commonly used for vaccinaton is S. typhi Ty21a. This strain is attenuated by a mutation in galE that inactivates a component essential in the galactose degradation pathway. In the meantime improved strains were developed recently, like the strain CVD 915, which is attenuated by a mutation in guaBA encoding an essential enzyme of the guanine synthesis pathway (Wang et al., 2001). Intraperitoneal application of the Ty21a strain that carried an expression plasmid encoding the nucleoprotein of measles virus as a DNA vaccine resulted in specific cytotoxic T cell responses (Fennelly et al., 1999). Low antibody responses have also been observed when ampicillin-treated mice were orally immunized with S. typhi Ty21a carrying a plasmid encoding a fragment of tetanus toxin (Woo et al., 2000). This demonstrates that the use of S. typhi as carrier for DNA vaccines is principally feasible. A comprehensive study was performed comparing nasally applied S. typhi CVD915 DguaBA carrying either a eukaryotic expression plasmid or a plasmid with an in vivo inducible bacterial promoter encoding an identical fragment of tetanus toxin as antigen (Pasetti et al., 1999). In addition, intramuscular injections of the naked eukaryotic expression plasmid were carried out in parallel experiments. With regard to antibody responses both immunizations involving bacteria were superior to direct applica-

DNA transfer by Salmonella

tion of the expression plasmid. Bacteria carrying the eukaryotic expression plasmid induced higher antibody levels then bacteria carrying the in vivo inducible prokaryotic expression plasmid under these conditions. Interestingly, despite the presence of antibodies against LPS after primary immunization, a booster reaction was observed using either of the two recombinant bacteria. Therefore, Salmonella-mediated DNA vaccination should also be possible in individuals that had encountered Salmonella before. The expression plasmid encoding the codonoptimized gp120 of HIV was used to compare the efficacy of different bacteria to act as carrier for mucosal DNA vaccination. Induction of T cell responses was taken as indicator. When similar numbers of S. flexneri, S. typhimurium or S. typhi were applied nasally to mice reasonable responses were obtained with S. flexneri and S. typhimurium as carriers, although S. flexneri appeared slightly better than S. typhimurium (Vecino et al., 2002). In this context, S. flexneri revealed an equal induction potential as the naked plasmid applied intramuscularly. Such comparisons although challenging should be accepted with some reservation. The physiology of such carrier bacteria is very different and nasal application is most likely not the route that will be applied in human studies. Recently, an additional member of the Salmonella spp. has proved to be functional as carrier for oral DNA vaccination. A vaccine strain of S. choleraesuis was shown to be able to transfer expression plasmids in vitro and in vivo (Shiau et al., 2001). Interestingly, in these experiments co-tranfer of two plasmids by the same bacterium was successfully attempted. One vector was based on a plasmid containing a ColE1 ori and encoding the glycoprotein C of the pseudorabies virus. The second vector contained a p15a ori and encoded prothymosin as an immunomodulatory cytokine to enhance the immune response. Primary peritoneal macrophages from mice could be shown to express both proteins after in vitro transfer using S. choleraesuis. Co-transfer in vivo resulted in superior induction of helper and killer T cells as well as enhanced antibody responses when compared with administration of Salmonella carrying only the glycoprotein C-encoding plasmid. Improved protection against a lethal dose of the pseudorabies virus could also be demonstrated after the mice had been vaccinated with bacteria that carried both plasmids as compared to bacteria carrying either one of them. These results suggest that most likely many different Salmonella strains can be used as DNA carrier. Thus the system of oral transgene vaccination most likely can be adapted to many different hosts by using the

99

appropriate invasive DNA carrier. In addition cotransfer of two plasmids containing compatible origins of replication offers a very simple and versatile way of modulating immune responses. DNA vaccination against model tumors in mice and rats using attenuated S. typhimurium Extremely promising results have been obtained using S. typhimurium-mediated oral DNA vaccination in tumor models. Employing b-gal as surrogate tumor antigen in an aggressive fibrosarcoma, partial protective immunity have been induced by orally administering Salmonella carrying b-gal-encoding plasmids (Paglia et al., 1998). Similarly, using the bgal-expressing murine renal cell carcinoma line RENCA-b-Gal, Zˆller and Christ (2001) have demonstrated superior efficacy in inducing tumor protection when the antigen-encoding plasmids were delivered orally by the Salmonella carrier as opposed to injecting naked DNA intramuscularly. These studies were extended to autologous tumor antigens, i.e antigens that are encoded by the tumor and might be also expressed in normal tissues of the animal. Thus, gp100 an autologous tumor antigen expressed in the murine melanoma line B16 was used for prophylactic vaccination (Cochlovius et al., 2002). Seventy percent of the mice were protected when challenged with tumor cells after several administrations of the tumor vaccine. Gp100-specific T cells could be detected in the spleen of such mice and by histology, antigen-expressing dendriticlike cells were found in the mesenteric lymph nodes shortly after intragastric administration of the recombinant Salmonella carrying the gp100-encoding plasmid. To improve the efficiency of such vaccines, gp100 was fused to the invariant chain that should target the antigen to compartments involved in the MHC class II presentation pathway. Additional IL-2 was administered subcutaneously around the tumor location in some of the experiments. The combinational treatment prolonged the survival of the mice and was most effective when the tumor was applied at the same time as the vaccination was started (Weth et al., 2001). Controversial to the above study, no prophylactic anti-tumor response could be induced in this work. Interestingly, when a yellow fluorescent protein was used as fusion partner for the invariant chain in mice receiving recombinant Salmonella, high numbers of protein-expressing cells comprising macrophages and dendritic cells were found in the PP and especially in the peritoneum. Using autologous tumor antigens even more promising results were obtained by the group of R.

100

S. Weiss

Reisfeld. First, they used Salmonella that carried minigenes encoding epitopes of the autologous tumor antigens gp100 and TRP2 fused to ubiquitin for immunization. This resulted in retardation of growth of the melanoma B16 (Xiang et al., 2000). Since gp100 and TRP2 are self-antigens this suggests that Salmonella-mediated DNA vaccination is able to break immunological tolerance towards autologous tumor antigens. These findings were extended to a murine neuroblastoma model where Salmonella-mediated DNA vaccination was performed with minigenes encoding epitopes of tyrosine hydroxylase, an autologous antigen found in this tumor. The minigene consisted of a T cell epitope fused to ubiquitin (Lode et al., 2000). Further extension of this work showed that also complete proteins without the ubiquitin fusion partner can be used as antigens. In a mouse that was transgenic for human carcinoembryonic antigen (hCEA), whereby hCEA functioned as self-antigen, oral immunization with S. typhimurium carrying an expression plasmid that encoded the complete hCEA, resulted in induction of specific cytotoxic T cells (Xiang et al., 2001a). In addition, the growth of a colon carcinoma expressing hCEA as tumor antigen was retarded. This response could be strongly improved by additionally administering intravenously a fusion protein of IL-2 with an antibody that targeted the recombinant cytokine to the tumor. When the same treatment was applied to a lung metastasis model using the same recombinant tumors and mice, most animals remained free of metastases (Niethammer et al., 2001a). A similar improvement of the protective immune response has been reported recently by the same group using the original B16 melanoma model described above and the epitopes of gp100 and TRP2 fused to ubiquitin as antigens. IL-2 targeted to the tumor was additionally applied as adjuvant shortly after tumor challenge. Targeting of IL-2 to the tumor was achieved via a fusion to an antiganglioside antibody, the antigen of which was expressed in the tumor. This treatment resulted in complete protection against the melanoma in most mice (Niethammer et al., 2001b). In these experiments it could also be shown that during the induction phase of the immune response no help from CD4 T cells was required to induce tumorspecific CD8 cytotoxic T cells. This is in agreement with the strong adjuvant capacity of the bacterial plasmid carrier. These studies culminated, so far, by including CD40 ligand (CD40L) as adjuvant. CD40L should support the activation and maturation of antigenpresenting cells. A fusion protein of a trimerizing

CD40L was generated with the hCEA described above. Salmonella-mediated DNA vaccination of mice transgenic for hCEA with the fusion construct of CD40L and hCEA was combined with the therapeutic injection of IL-2 targeted to the tumor in vivo as described above. Mice treated this way were completely protected against a tumor challenge by a chemically induced colon adenocarcinoma line that expressed the hCEA as transgene (Xiang et al., 2001b). The application of any component of this combination alone only partially protected the mice against a challenge with the same tumor. However, a rather unexpected result was obtained when Salmonella-mediated DNA vaccination was applied to a splice variant of CD44 ± CD44v2 ± in a rat tumor model (Zˆller, 2002). CD40v2 is a selfantigen although it is mainly expressed during fetal development. The tumor employed, a lymphoma, was therefore rendered transgenic for the CD44v2. Cytotoxic and helper T cell responses could be measured after intragastric administration of the recombinant Salmonella. However, the tumor grew faster in vaccinated mice and metastases appeared in the thymus. Histological and cytological analyses revealed expression of the antigen in peritoneal myeloid cells similar to the results reported above. Surprisingly, however, a few antigen-expressing myeloid cells could also be detected in the thymus. Since CD44 ± and CD44v2 in particular ± might be a thymic homing factor this might explain why CD44v2-expressing myeloid cells as well as CD44v2-expressing tumor cells can be found in the thymus. Unsatisfactorily, however, is the explanation why only the vaccinated mice exhibited thymic metastases. Tolerance induction against the CD44v2 was observed, at least the specific response that was originally observed after the initial administrations declined. The mechanism of such a tolerance induction remains unexplained.

Salmonella-mediated gene therapy The obvious efficiency of S. typhimurium in transferring eukaryotic expression plasmids to host cells in vivo and in vitro also has prompted its use for applications different than genetic vaccination. Human dendritic cells have been shown to be susceptible to transfection by Salmonella (Dietrich et al., 2001) although the percentage of transfectants obtained is still low. Our original finding that primary macrophages can be transfected to a high degree in vitro using S. typhimurium (Darji et al., 1997) has been further extended. Percentages of

DNA transfer by Salmonella

transfectants close to 100% have been obtained with murine as well as human macrophages (Paglia et al., 2000; Montosi et al., 2000). The latter system was used to complement a monogenic defect in macrophages from patients with hereditary hemochromatosis by transferring a plasmid encoding the cDNA of the hemochromatosis gene HFE (Montosi et al., 2000). In addition, several applications in vivo have been reported. Mice defective in IFN-g would normally succumb to a challenge with S. typhimurium aroA. However, when these bacteria carried an expression plasmid encoding IFN-g and thus were able to transfer DNA that leads to a complementation of the genetic defect at least in some cells, the mice were able to resist the bacterial challenge (Paglia et al., 2000). Similarly, transfer of human or murine IL-12- and GM-CSF-encoding cDNA via S. typhimurium resulted in measurable levels of the particular recombinant cytokine in the serum of such mice and caused the retardation of subcutaneously applied tumors (Yuhua et al., 2001). In a murine B cell lymphoma model, Salmonella were used to transfer a plasmid encoding a soluble form of the human CD40 ligand orally (Urashima et al., 2000). Stimulation of normal antigen-presenting cells with CD40L results in up-regulation of MHC class I and class II as well as co-stimulatory molecules. In the particular B cell lymphomas employed in these experiments, stimulation via CD40L leads to growth suppression in vitro and in vivo. Oral administration of the recombinant S. typhimurium protected mice against a simultaneous challenge with the B cell lymphoma. This treatment was still partially effective when the recombinant Salmonella were applied one week after the challenge and still retarded the tumor growth when administered two to three weeks after tumor application. CD40L was detectable for several weeks in the serum of such mice. Histological examination demonstrated that after oral administration of the recombinant Salmonella many cells expressed CD40L in PP while only few of such cells could be found in spleen. These findings convincingly demonstrate the high potential of Salmonella spp. as carrier for DNA vaccines and gene therapy.

Possible mechanism of induction of immune responses by Salmonellamediated oral DNA vaccination In the original work (Darji et al., 1997) responses against the transgene in all three specific compart-

101

ments of the immune system were described. Cytotoxic and helper T cells as well as antibodies could be demonstrated after oral administration of the recombinant attenuated Salmonella. In the meantime it is clear from our own work and from others that this type of DNAvaccination is extremely efficient in raising T cell response but antibody responses are induced only under optimal conditions (see above). Therefore, it is essential to understand the mechanisms acting during Salmonella-mediated mucosal DNA vaccination in order to be able to optimize this system. It is generally accepted that after oral uptake attenuated Salmonella breach the intestinal barrier mainly via M cells found in the epithelium overlaying the PP. Macrophages and dendritic cells (DC) are associated with the apical site of such cells and thus the bacteria are probably immediately phagocytosed by these cells. Salmonella residing in DC within the dome area of PP have been demonstrated (Hopkins et al., 2000). In these phagocytes the bacteria will start to replicate and die possibly due to their metabolic attenuation. This should result in the release of their plasmids and the in vivo transfection of the infected cells which in turn will produce the antigen. High numbers of antigenexpressing cells have been found in the PP (Urashima et al., 2000). At the same time, the bacterial infection activates the phagocytic cells resulting in different activities. DC might be induced to migrate into other regions of the PP or to the draining lymph nodes. Antigen-expressing cells with DC or macrophage shape have been found in the mesenteric lymph nodes and a few in the spleen (Cochlovius et al., 2002; Urashima et al., 2000). We find it unlikely that such cells were transfected in situ by disseminating Salmonella since we never could detect viable plasmid-carrying bacteria in any organ even when tested shortly after application. In addition, treatment of mice with gentamycin an antibiotic that remains outside the cells and the gut lumen, did not influence the induction of an immune response. Thus, we find it unlikely that attenuated Salmonella carrying the eukaryotic expression plasmid reach any tissue other than the PP. However, surprisingly high numbers of antigen-expressing myeloid cells have also been detected in the peritoneal cavity after Salmonella-mediated DNA vaccination (Weth et al., 2001; Zˆller, 2002). Whether the peritoneal cells have been directly transfected by disseminating recombinant Salmonella (but see above) or whether cells have migrated from the intestinal tissue into the peritoneum is unclear. The above described scenario only would explain the induction of cytotoxic MHC class I-restricted

102

S. Weiss

responses since most of the antigens that have been used remained in the cytosol of the antigen-presenting cell and were not secreted. Nevertheless, MHC class II-dependent T cell responses and antibody responses have been found to be induced by this way of immunization. Therefore, other explanations are needed. It is know that Salmonella upon infection of activated macrophages induce programmed cell death in these infected cells (Chen et al., 1996; Monack et al., 1996). Two types of cell death were discovered. One acts immediately and is mainly induced by virulence factors of pathogenicity island I (Hersh et al., 1999). It is strongly dependent on the cytosolic protease caspase I of the host cell. This type of induced cell death is essential for the dissemination of Salmonella from the PP to the deep organs (Monack et al., 2000). Mice defective in caspase I are highly resistant to oral Salmonella infections and the pathogen cannot disseminate from the PP. A second mechanism exists which requires 24 hours to become active and is mainly dependent on virulence factors of pathogenicity island II but also partly requires caspase I (Monack et al., 2001). While still little is known about the delayed cell death the immediate type has been studied in detail. The bacterial virulence factor responsible for this was identified as SipB which is injected into the cytosol of the host cell via the type III secretion system of pathogenicity island I (Hersh et al., 1999). Its target molecule, caspase I, is known to activate IL-1b and IL-18 for secretion. Actually IL-1b has been demonstrated to be secreted by Salmonella-infected macrophages (Monack et al., 2001). However, caspase I is not known to activate the common apoptotic pathways. In agreement with this is that the programmed cell death after infection by Salmonella shares features of apoptosis as well as of necrosis (Brennan and Cookson, 2000). Pyroptosis was therefore suggested as a descriptive term for this type of cell death (Cookson and Brennan, 2001). While cells that undergo apoptosis remain largely intact during this process, cellular components are found to leak out from the cell interior during necrosis. Intracellular components have been demonstrated in the supernatant from macrophages infected by Salmonella and undergoing programmed cell death (Brennan and Cookson, 2000). Thus it is possible that after Salmonella-mediated oral DNA vaccination intracellular antigens are released from infected macrophages and might be acquired by neighbouring DC which could present the antigen via MHC class II but also via MHC class I as described above. Also antibody production could be explained this way. More likely but not mutually

exclusive, however, is the possibility that neighbouring DC take up the complete dying macrophages and re-present the antigens expressed by this cell. This phenomenon is known as cross-presentation. Cross-presentation was discovered when mice were immunized with cells from another mouse strain that was different in the major as well as the minor histocompatibility antigens. The response against the minor antigens was found to be restricted by the MHC molecules of the immunized animals (Bevan et al., 1976). Thus, a transfer of components of donor cells or peptides must have taken place. Some controversy on the importance of crosspresentation in vivo still exists (Zinkernagel, 2002). Nevertheless, it is widely accepted that cross-presentation is essential for the induction of immune responses against pathogens that do not directly infect dendritic cells since dendritic cells are believed to be the only antigen-presenting cells that are able to activate naive resting T cells (reviewed in (Heath and Carbone, 2001a, b)). On the other hand cross-presentation might also constitutively take place under normal conditions. Thus, dendritic cells in the mesenteric lymph nodes have been observed that contain epithelial cells from the gut (Huang et al., 2000). Similarly, dendritic cells were found to present transgenic antigen from tissues under normal circumstances and might therefore also contribute to the maintenance of the immunological tolerance ((Kurts et al., 1997) and reviewed in (Heath and Carbone, 2001a, b)). The subpopulation of dendritic cells that is mainly responsible for crosspresentation was identified as the so-called lymphoid dendritic cell that is characterized by the surface markers CD11c, CD8 and DEC205 (den Haan et al., 2000; Pooley et al., 2001). However, more recently it became clear that also myeloid DC with the markers CD11c and CD11b can crosspresent antigen given the right activation (den Haan et al., 2002). The involvement of cross-presentation during the induction of an immune response by mucosal Salmonella-mediated DNA vaccination would fit well with our data (Darji et al., unpublished). When we tested the antigen-presentation capacity of macrophages and dendritic cells from PP and spleen shortly after Salmonella-mediated oral DNA vaccination we noticed that macrophages in the PP could present antigen only via MHC class I while dendritic cells could present antigen via MHC class I and II. Since we used b-gal a non-secreted antigen that should be presented only via MHC class I, crosspresentation was the most likely explanation for this phenomenon. By cell sorting we could show that the population of dendritic cells that carried the marker

DNA transfer by Salmonella

DEC205 was involved. In addition, we could show that in the spleen antigen was not presented by macrophages at all, but in the early phase some dendritic cells could present only via class I and later presentation via class I and class II could be detected. Again, DC of a DEC205-expressing subpopulation were taking part in this. Since we could never observe plasmid-carrying bacteria under these conditions, we interpret these findings that macrophages and dendritic cells are infected in the PP by the plasmid-carrying Salmonella. After death of the Salmonella and in vivo transfection antigen is expressed and presented via MHC class I by such cells. At the same time some of them undergo programmed cell death induced by the Salmonella infection. Such cells are taken up by the lymphoid dendritic cells, and antigens expressed by the dying cells are re-presented via MHC class I and II. These cross-presenting DC also are responsible for the transport of the antigens to the deep lymphoid organs as could be shown by inhibition of antigen transport by treatment with anti-DEC205 antibodies. When migrating via the lymph such cells should not pass the draining i.e. the mesenteric lymph node. Therefore, the dissemination of the antigen might also take place via the blood stream. An alternative and again not mutual exclusive explanation for the induction of systemic immune responses after Salmonella-mediated DNA vaccination was suggested by the recent finding that dendritic cells in the lamina propria can extend dendrites into the gut lumen and use this to sample antigen (Rescigno et al., 2001). Thus, such dendritic cells could also sample recombinant expression plasmid carrying Salmonella. After being transfected due to the lysis of the attenuated bacteria they could transport the antigen to the lymphoid organs and induce the immune response.

Conclusion and outlook The results reviewed here were obtained with the first generation of the Salmonella-mediated mucosal DNA transfer system. Already from these data its great potential becomes obvious. Successful gene therapy was carried out in model systems, and from the experiments using tumor proteins it can be concluded that tolerance against tumor-associated antigens can be broken with Salmonella as carrier for DNA vaccines. On the other hand some problems became apparent. At the moment the induction of an antibody response is only possible under optimal conditions and is mainly systemic and not mucosal

103

as might have been expected for a mucosally applied vaccine. Part of the problem might be that the Salmonella aroA strain that is currently predominantly used is dying very quickly when containing high-copy-number plasmids (Garmory et al., 2002) and thus only few cells might be transfected or only few plasmid molecules are transferred due to the same reason. The strains used at the moment might also be too efficient in inducing programmed cell death of the cells they have infected, again limiting the antigen expression by that. However, stability of the vector system seems to be one of the biggest problems at the moment. The plasmids are usually stabilized by an ampicillin resistance marker which encodes the secretory b-lactamase. But at the same time the high-copy-number plasmids that are usually employed most likely also influence the stability in culture and in vivo (Garmory et al., 2002). Thus several optimization steps will be possible to increase the level of antigen expression after Salmonella-mediated DNA vaccination. This (combined with the ease of preparation and storage as well as with the general acceptance of an oral application in combination with approved vaccine strains) should render this system highly versatile for all kinds of vaccination problems. Acknowledgement. The author is grateful to H. Loessner for critical reading of the manuscript, to B. Zimmermann for secretarial assistance and to S. zur Lage and A. Darji for providing unpublished data. This work was supported in part by grants from the DFG and the BMBF.

References Al-Mariri, A., Tibor, A., Lestrate, P., Mertens, P., De Bolle, X., Letesson, J.-J.: Yersinia enterocolitica as a vehicle for naked DNA vaccine encoding Brucella abortus bactoferritin or P39 antigen. Infect. Immun. 70, 1915 ± 1923 (2002). Bevan, M. J.: Cross-priming for a secondary cytotoxic response to minor H antigens with H-2 congenic cells which do not cross-react in the cytotoxic assay. J. Exp. Med. 143, 1283 ± 1288 (1976). Brennan, M. A., Cookson, B. T.: Salmonella induces macrophage death by caspase-1-dependent necrosis. Mol. Microbiol. 38, 31 ± 40 (2000). Brunham, R. C., Zhang, D.: Transgene as vaccine for Chlamydia. Am. Heart J. 138, 519 ± 522 (1999). Catic, A., Dietrich, G., Gentschev, I., Goebel, W., Kaufmann, S. H., Hess, J.: Introduction of protein or DNA delivered via recombinant Salmonella typhimurium into the major histocompatibility complex class I presentation pathway of macrophages. Microbes Infect. 1, 113 ± 121 (1999).

104

S. Weiss

Charpentier, E., Gerbaud, G., Courvalin, P.: Conjugative mobilization of the rolling-circle plasmid pIP823 from Listeria monocytogenes BM4293 among gram-positive and gram-negative bacteria. J. Bacteriol. 181, 3368 ± 3374 (1999). Chen, L., Kaniga, K., Gala¬n, J. E.: Salmonella spp. are cytotoxic for cultured macrophages. Mol. Microbiol. 21, 1101 ± 1115 (1996). Cochlovius, B., Stassar, M. J. J. G., Schreurs, M. W., Brenner, A., Gosse, J. A.: Oral DNA vaccination: antigen uptake and presentation by dendritic cells elicits protective immunity. Immunol. Lett. 80, 89 ± 96 (2002). Cookson, B. T., Brennan, M. A.: Pro-inflammatory programmed cell death. Trends Microbiol. 9, 113 ± 114 (2001). Courvalin, P., Goussard, S., Grillot-Courvalin, C.: Gene transfer from bacteria to mammalian cells. C. R. Acad. Sci. III 318, 1207 ± 1212 (1995). Darji, A., Guzman, C. A., Gerstel, B., Wachholz, P., Timmis, K. N., Wehland, J., Chakraborty, T., Weiss, S.: Oral somatic transgene vaccination using attenuated S. typhimurium. Cell 91, 765 ± 775 (1997). Darji, A., zur Lage, S., Garbe, A. I., Chakraborty, T., Weiss, S.: Oral delivery of DNA vaccines using attenuated Salmonella typhimurium as carrier. FEMS Immunol. Med. Microbiol. 27, 341 ± 349 (2000). den Haan, J. M. M., Bevan, M. J.: Constitutive versus activation-dependent cross-presentation of immune complexes by CD8‡ and CD8 dendritic cells in vivo. J. Exp. Med. 196, 817 ± 827 (2002). den Haan, J. M. M., Lehar, S. M., Bevan M. J.: CD8‡ but not CD8 dendritic cells cross-prime cytotoxic Tcells in vivo. J. Exp. Med. 12, 1685 ± 1695 (2000). Dietrich, G., Bubert, A., Gentschev, I., Sokolovic, Z., Simm, A., Catic, A., Kaufmann, S. H., Hess, J., Szalay, A. A., Goebel, W.: Delivery of antigen-encoding plasmid DNA into the cytosol of macrophages by attenuated suicide Listeria monocytogenes. Nat. Biotechnol. 16, 181 ± 185 (1998). Dietrich, G., Kolb-M‰urer, A., Spreng, S. Schartl, M., Goebel, W., Gentschev, I.: Gram-positive and gramnegative bacteria as carrier systems for DNA vaccines. Vaccine 19, 2506 ± 2512 (2001). Dietrich, G., Spreng, S., Gentschev, I., Goebel, W.: Bacterial systems for the delivery of eukaryotic antigen expression vectors. Antisense Nucleic Acid Drug Dev. 10, 391 ± 399 (2000). Fennelly, G. J., Khan, S. A., Abadi, M. A., Wild, T. F., Bloom, B. R.: Mucosal DNA vaccine immunization against measles with a highly attenuated Shigella flexneri vector. J. Immunol. 162, 1603 ± 1610 (1999). Flo¬, J., Tisminetzky, S., Baralle, F.: Oral transgene vaccination mediated by attenuated Salmonellae is an effective method to prevent Herpes simplex virus-2 induced disease in mice. Vaccine 19, 1772 ± 1782 (2001). Gagnon, E., Duclos, S., Rondeau, C., Chevet, E., Cameron, P. H., Steele-Mortimer, O., Paiement, J.,

Bergeron, J. M., Desjardins, M.: Endoplasmic reticulum-mediated phagocytosis is a mechanism of entry into macrophages. Cell 110, 119 ± 131 (2002). Garmory, H. S., Griffin, K. F., Leary, S. E. C., Perkins, S. D., Brown, K. A., Titball, R. W.: The effect of recombinant plasmids on in vivo colonisation of Salmonella enterica serovar Typhimurium strains is not reflected by in vitro cellular invasion assays. Vaccine 20, 3239 ± 3243 (2002). Gentschev, I., Sokolovic, Z., Mollenkopf, H. J., Hess, J., Kaufmann, S. H., Kuhn, M., Krohne, G. F., Goebel, W.: Salmonella strain secreting active listeriolysin changes its intracellular localization. Infect. Immun. 63, 4202 ± 4205 (1995). Grillot-Courvalin, C., Goussard, S., Courvalin, P.: Wildtype intracellular bacteria deliver DNA into mammalian cells. Cell. Microbiol. 4, 177 ± 186 (2002). Grillot-Courvalin, C., Goussard, S., Huetz, F., Ojcius, D. M., Courvalin, P.: Functional gene transfer from intracellular bacteria to mammalian cells. Nat. Biotechnol. 16, 862 ± 866 (1998). Heath, W. R., Carbone, F. R.: Cross-presentation, dendritic cells, tolerance and immunity. Annu. Rev. Immunol. 19, 47 ± 64 (2001a). Heath, W. R., Carbone, F. R.: Cross-presentation in viral immunity and self-tolerance. Nat. Rev. Immunol. 1, 126 ± 135 (2001b). Heinemann, J. A., Sprague, G. F., Jr.: Bacterial conjugative plasmids mobilize DNA transfer between bacteria and yeast. Nature 340, 205 ± 209 (1989). Hense, M., Domann, E., Krusch, S., Wachholz, P., Dittmar, K. E. J., Rohde, M., Wehland, J., Chakraborty, T., Weiss, S.: Eukrayotic expression plasmid transfer from the intracellular bacterium Listeria monocytogenes to host cells. Cell. Microbiol. 3, 599 ± 609 (2001). Hersh, D., Monack, D. M., Smith, M. R., Ghori, N., Falkow, S., Zychlinski, A.: The Salmonella invasin SipB induces macrophage apoptosis by binding to caspase-1. Proc. Natl. Acad. Sci. USA 98, 2396 ± 2401 (1999). Hopkins, S. A., Niedergang, F., Corthesy-Theulaz, I. E., Kraehenbuhl, J.-P.: A recombinant Salmonella typhimurium vaccine strain is taken up and survives within murine Peyer's patch dendritic cells. Cell. Microbiol. 2, 59 ± 68 (2000). Huang, F. P., Platt, N., Wykes, M., Major, J. R., Powell, T. J., Jenkins, C. D., MacPherson, G. G.: A discrete subpopulation of dendritic cells transports apoptotic intestinal epithelial cells to T cell areas of mesenteric lymph nodes. J. Exp. Med. 191, 435 ± 444 (2000). Jain, V., Mekalanos, J. J.: Use of lambda phage S and R gene products in an inducible lysis system for Vibrio cholerae and Salmonella enterica serovar typhimurium-based DNA vaccine delivery systems. Infect. Immun. 68, 986 ± 989 (2000). Kunik, T., Tzfira, T., Kapulnik, Y., Gafni, Y., Dingwall, C., Citovsky, V.: Genetic transformation of HeLa cells by Agrobacterium. Proc. Natl. Acad. Sci. USA 98, 1871 ± 1876 (2001).

DNA transfer by Salmonella Kurts, C., Kosaka, H., Carbone, F. R., Miller, J. F. A. P., Heath, W. R.: Class I-restricted cross-presentation of exogenous self-antigens leads to deletion of autoreactive CD8(‡). J. Exp. Med. 186, 239 ± 245 (1997). Lessl, M., Lanka, E.: Common mechanisms in bacterial conjugation and Ti-mediated T-DNA transfer to plant cells. Cell 77, 321 ± 324 (1994). Lode, H. N., Pertl, U., Xiang, R., Gaedicke, G., Reisfeld, R. A.: Tyrosine hydroxylase-based DNA vaccination is effective against murine neuroblastoma. Med. Pediatr. Oncol. 35, 641 ± 646 (2000). Lubitz, W.: Bacterial ghosts as carrier and targeting systems. Expert Opin. Biol. Ther. 5, 765 ± 771 (2001). Mastroeni, P., Chabalgoity, J. A., Dunstan, S. J., Maskell, D. L., Dougan, G.: Salmonella: immune responses and vaccines. Vet. J. 161, 132 ± 164 (2001). Monack, D. M., Detweiler, C. S., Falkow, S.: Salmonella pathogenicity island 2-depedent macrophage death is mediated in part by the host cysteine protease caspase-1. Cell. Microbiol. 3, 825 ± 837 (2001). Monack, D. M., Hersh, D., Ghori, N. Bouley, D., Zychlinsky, A., Falkow, S.: Salmonella exploits caspase-1 to colonize Peyer's patches in a murine typhoid model. J. Exp. Med. 192, 249 ± 258 (2000). Monack, D. M., Raupach, B., Hromockyi, A. E., Falkow, S.: Salmonella typhimurium invasion induces apoptosis in infected macrophages. Proc. Natl. Acad. Sci. USA 93, 9833 ± 9838 (1996). Montosi, G., Paglia, P., Garuti, C., Guzma¬n, C. A., Bastin, J. M. Colombo, M. P., Pietrangelo, A.: Wildtype HFE protein normalizes transferrin iron accumulation in macrophages from subjects with hereditary hemochromatosis. Blood 96, 1125 ± 1129 (2000). Niethammer, A. G., Primus, F. J., Xiang, R., Dolman, C. S., Ruehlmann, J. M., Ba, Y., Gillies, S. D., Reisfeld, R. A.: An oral DNA vaccine against human carcinoembryonic antigen (CEA) prevents growth and dissemination of Lewis lung carcinoma in CEA transgenic mice. Vaccine 20, 421 ± 429 (2001a). Niethammer, A. G., Xiang, R., Ruehlmann, J. M., Lode, H. N. Dolman, C. S., Gillies, S. D., Reisfeld R. A.: Targeted Interleukin 2 therapy enhances protective immunity induced by an autologous oral DNA vaccine against murine melanoma. Cancer Res. 61, 6178 ± 6184 (2001b). Norbury, C. C., Hewlett, L. J., Prescott, A. R., Shastri, N., Watts, C.: Class I MHC presentation of exogenous soluble antigen via macropinocytosis in bone marrow macrophages. Immunity 3, 783 ± 791 (1995). Paglia, P., Medina, E., Arioli, I., Guzman, C. A., Colombo, M. P.: Gene transfer in dendritic cells, induced by oral DNA vaccination with Salmonella typhimurium, results in protective immunity against a murine fibrosarcoma. Blood 92, 3172 ± 3176 (1998). Paglia, P., Terrazzini, N., Schulze, K., Guzman, C. A., Colombo, M. P.: In vivo correction of genetic defects of monocyte/macrophages using attenuated Salmonella as oral vectors for targeted gene delivery. Gene Ther. 7, 1725 ± 1730 (2000).

105

Pasetti, M. F., Anderson, R. J., Noriega, F. R., Levine, M. M., Sztein, M. B.: Attenuated DguaBA Salmonella typhi vaccine strain CVD 915 as a live vector utilizing prokaryotic or eukaryotic expression systems to deliver foreign antigens and elicit immune responses. Clin. Immunol. 92, 76 ± 89 (1999). Pooley, J. L, Heath, W. R., Shortman, K.: Cutting edge: intravenous soluble antigen is presented to CD4 cells, but cross-presented to CD8 T cells by CD8‡ dendritic cells. J. Immunol. 166, 5327 ± 5330 (2001). Powell, R. J., Lewis, G. K., Hone, D. M.: Introduction of eukaryotic expression cassettes into animal cells using bacterial vector delivery system. In: Vaccines 96: Molecular approaches to the control of infectious disease. pp. 183 ± 187. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1996. Reinhardt, R. L., Khoruts, A., Mercia, R., Zell, T., Jenkins, M. K.: Visualizing the generation of memory CD4 T cells in the whole body. Nature 410, 101 ± 105 (2001). Rescigno, M., Urbano, M., Valzasina, B., Francolini, M., Rotta, G., Bonasio, R., Granucci, F., Kraehenbuhl, J.P., Ricciardi-Castagnoli, P.: Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nat. Immunol. 2, 361 ± 367 (2001). Rodriguez, A., Regnault, A., Kleijmeer, M., RicciardiCastagnoli, P., Amigorena, S.: Selective transport of internalized antigens to the cytosol for MHC class I presentation in dendritic cells. Nat. Cell Biol. 1, 362 ± 368 (1999). Schaffner, W.: Direct transfer of cloned genes from bacteria to mammalian cells. Proc. Natl. Acad. Sci. USA 77, 2163 ± 2167 (1980). Shata, M. T., Reitz, M. S., Jr., DeVico, A. L., Lewis, G. K., Hone, D. M.: Mucosal and systemic HIV-1 Env-specific CD8‡ T-cells develop after intragastric vaccination with a Salmonella Env DNA vaccine vector. Vaccine 20, 623 ± 629 (2001). Shiau, A.-L., Chen, Y.-L., Liao, C.-Y., Huang, Y.-S., Wu, C.-L.: Prothymosin a enhances protective immune responses induced by oral DNA vaccination against pseudorabies delivered by Salmonella choleraesuis. Vaccine 19, 3947 ± 3956 (2001). Sizemore, D. R., Branstrom, A. A., Sadoff, J. C.: Attenuated Shigella as a DNA delivery vehicle for DNAmediated immunization. Science 270, 299 ± 302 (1995). Trieu-Cuot, P., Derlot, E., Courvalin, P.: Enhanced conjugative transfer of plasmid DNA from Escherichia coli to Staphylococcus aureus and Listeria monocytogenes. FEMS Microbiol. Lett. 109, 19 ± 23 (1993). Urashima, M., Suzuki, H., Yuza, Y., Akiyama, M., Ohno, N., Eto, Y.: An oral CD40 ligand gene therapy against lymphoma using attenuated Salmonella typhimurium. Blood 95, 1258 ± 1263 (2000). Vecino, W. H., Morin, P. M., Agha, R., Jacobs, W. R., Jr., Fennelly, G. J.: Mucosal DNA vaccination with highly attenuated Shigella is superior to attenuated

106

S. Weiss

Salmonella and comparable to intramuscular DNA vaccination for T cells against HIV. Immunol. Lett. 82, 197 ± 204 (2002). Wang, J. Y., Pasetti, M. F., Noriega, F. R., Anderson, R. J., Wasserman, S. S., Galen, J. E., Sztein, M. B., Levine, M. M.: Construction, genotypic and phenotypic characterization, and immunogenicity of attenuated DguaBA Salmonella enterica serovar Typhi strain CVD 915. Infect. Immun. 69, 4734 ± 4741 (2001). Weth, R., Christ, O., Stevanovic, S., Zˆller, M.: Gene delivery by attenuated Salmonella typhimurium: Comparing the efficacy of helper versus cytotoxic T cell priming in tumor vaccination. Cancer Gene Ther. 8, 599 ± 611 (2001). Woo, P. C., Tsoi, H. W., Leung, H. C., Wong, L. P., Wong, S. S., Chan, E., Yuen, K. Y.: Enhancement by ampicillin of antibody responses induced by a protein antigen and a DNA vaccine carried by live-attenuated Salmonella enterica serovar Typhi. Clin. Diagn. Lab. Immunol. 7, 596 ± 599 (2000). Woo, P. C. Y., Wong, L. P., Zheng, B. J., Yuen, K. Y.: Unique immunogenicity of hepatitis B virus DNA vaccine presented by live-attenuated Salmonella typhimurium. Vaccine 19, 2945 ± 2954 (2001). Xiang, R., Lode, H. N., Chao, T. H., Ruehlmann, J. M., Dolman, C. S., Rodriguez, F., Whitton, J. L., Overwijk, W. W., Restifo, N. P., Reisfeld, R. A.: An autologous oral DNA vaccine protects against murine melanoma. Proc. Natl. Acad. Sci. USA 97, 5492 ± 5497 (2000). Xiang, R., Silletti, S., Lode, H. N., Dolman, C. S., Ruehlmann, J. M., Niethammer, A. G., Pertl, U.,

Gillies, S. D., Primus, F. J, Reisfeld, R. A.: Protective immunity against human carcinoembryonic antigen (CEA) induced by an oral DNA vaccine in CEAtransgenic mice. Clin. Cancer Res. 7, 865S ± 864S (2001a). Xiang, R., Primus, F. J., Ruehlmann, J. M., Niethammer, A. G., Silletti, S., Lode, H. N, Dolman, C. S., Gillies, S. D., Reisfeld, R. A.: A dual-function DNA vaccine encoding carcinoembryonic antigen and CD40 ligand trimer induces T cell-mediated protective immunity against colon cancer in carcinoembryonic antigentransgenic mice. J. Immunol. 167, 4560 ± 4565 (2001b). Yuhua, L., Y. Kunyuan, G., Hui, C., Yongmei, X., Chaoyang, S., Xun, T., Daming, R.: Oral cytokine gene therapy against murine tumor using attenuated Salmonella typhimurium. Int. J. Cancer 94, 438 ± 443 (2001). Zheng, B., Woo, P. C. Y., Ng, M., Tsoi, H., Wong, L., Kwok-Yung, Y.: A crucial role of macrophages in the immune responses to oral DNA vaccination against hepatitis B virus in a murine model. Vaccine 20, 140 ± 147 (2001). Zinkernagel, R. M.: On cross-priming of MHC class-I specific CTL: rule or exception? Eur. J. Immunol. 32, 2385 ± 2392 (2002). Zˆller, M., Christ, O.: Prophylactic tumor vaccination: comparison of effector mechanisms initiated by protein versus DNA vaccination. J. Immunol. 166, 3440 ± 3450 (2001). Zˆller, M.: Unexpected induction of unresponsiveness by vaccination with transformed Salmonella typhimurium. J. Immunol. 25, 162 ± 175 (2002).