The present invention is directed to an attenuated Salmonella comprising a eukaryotic expression vector for delivery of the eukaryotic expression vector to a eukaryotic cell. Delivery may be to eukaryotic cells cultured in vitro or to cells in vivo, such as by oral administration of the attenuated Salmonella comprising the eukaryotic expression vector.

SUMMARY OF THE INVENTION

An attenuated strain of Salmonella typhimurium has been used as a vehicle for oral genetic immunization. Eukaryotic expression vectors containing the genes for b-galactosidase, or truncated forms of ActA and listeriolysin--two virulence factors of Listeria monocytogenes--that were controlled by an eukaryotic promoter have been used to transform a S. typhimurium aroA strain. Multiple or even single immunizations with these transformants induced a strong cytotoxic and helper T cell response as well as an excellent antibody response. Multiple immunizations with listeriolysin transformants protected the mice completely against a lethal challenge of L. Monocytogenes. Partial protection was already observed with a single dose. ActA appeared not to be a protective antigen.

The strength and the kinetics of the response suggested that the heterologous antigens were expressed within the eukaryotic host cells following transfer of plasmid DNA from the bacterial carrier strain. Transfer of plasmid DNA could be unequivocally shown in vitro using primary peritoneal macrophage. The demonstration of RNA splice products and expression of .beta.-galactosidase in the presence of tetracycline--an inhibitor of bacterial protein synthesis--indicated that the gene was expressed by host cells rather than bacteria. Oral genetic immunization with Salmonella carriers provides a highly versatile system for antigen delivery, represents a potent system to identify candidate protective antigens for vaccination, and will permit efficacious generation of antibodies against virtually any DNA segment encoding an open reading frame.

According to one embodiment the invention concerns an attenuated Salmonella strain carrying an eucaryotic expression vector for the expression of a heterologous gene or gene fragment or an autologous gene or gene fragment comprised by the vector within an open reading frame, wherein the attenuation is adjusted to a vaccination of vertebrates including humans.

The Salmonella strain according to the invention can be a S. typhimurium strain, especially S. typhimurium aroA SL 7207 or S. typhimurium LT2 and preferably aroA544 (ATCC 335).

Further, the Salmonella strain according to the invention can be a S. typhi strain, especially S. typhi Ty21a.

According to the invention Salmonella strains are comprised, wherein the eucaryotic expression vector is or can be derived from the known plasmid pCMV.beta. which comprises the structural gene of .beta.-galactosidase (.beta.-gal) under the control of the human cytomegalovirus (CMV) immediate early promoter comprised by the plasmid pCMV.beta. per se and includes a splice donor, two splice acceptor sites in between the promoter and the .beta.-galactosidase gene, and facultatively the polyadenylation site of SV40.

The Salmonella strain according to the invention can be characterized by a heterologous gene or an autologous gene coding for a protein and especially an immunogenic protein or protective antigen.

According to the invention Salmonella strains are comprised wherein the heterologous gene is selected from the group consisting of the Escherichia coli-.beta.-galactosidase gene (lacZ-gene), a non-hemolytic truncated variant of the Listeria monocytogenes--listeriolysine gene (hly gene) and a truncated variant of the Listeria monocytogenes--actA gene (actA gene).

Another embodiment of the invention concerns a vaccine for oral, nasal, mucosal, intravenous, intraperitonal, intradermal, or subcutaneous gene delivery to vertebrates including humans, wherein the vaccine comprises a Salmonella strain according to the invention.

Further, another embodiment of the invention concerns a use of a Salmonella strain according to the invention or of a vaccine according to the invention for expression screening of heterologous genomic DNA libraries or genomic cDNA libraries by DNA vaccination in vertebrates including humans.

Finally, another embodiment of the invention concerns a process for the recovery of (i) an attenuated Salmonella strain carrying an eucaryotic expression vector for the expression of a heterologous gene or gene fragment or an autologous gene or gene fragment comprised by the vector within an open reading frame, wherein the attenuation is adjusted to a vaccination of vertebrates including humans; or (ii) a vaccine for oral, nasal, mucosal, intravenous, intraperitonal, intradermal, or subcutaneous gene delivery to vertebrates including humans, wherein the vaccine comprises a Salmonella strain according to (i) or (iii) an immunogenic protein or protective antigen as expression product of an eucaryotic expression vector according to (i), characterized by (a) using genetic information from a heterologous or autologous DNA or cDNA library as gene fragment or gene to be expressed by an eucaryotic expression vector carried by an attenuated Salmonella strain, wherein the attenuation is adjusted to a vaccination of vertebrates including humans, (b) carrying out a DNA vaccination by means of the attenuated Salmonella strain according to (a) in a vertebrate or human being, (c) carrying out an expression screening for an expression product of a gene or gene fragment according to (a) providing an immune response (d) and recovering a Salmonella strain according to the invention or of a vaccine according to the invention or of an immunogenic protein or protective antigen providing an immune response in vertebrates including humans.

To sum up, we report that orally administered S. typhimurium aroA carrying plasmids encoding .beta.-galactosidase (.beta.-gal) of Escherichia coli, or truncated forms of ActA or listeriolysin of Listeria monocytogenes under the control of an eukaryotic promoter induce an efficient humoral and cellular immune response. The strength and kinetics of the response is only compatible with the interpretation of a transfer of the expression plasmid from the Salmonella carrier to the nucleus of APC of the host. .beta.-galactosidase activity was detectable even five weeks after administration of the oral vaccine. In addition, in vitro experiments with mouse primary macrophages demonstrated an efficient transfer of plasmid DNA from attenuated bacteria into the nucleus of phagocytic host cells.

Results

To achieve genetic immunization with a live attenuated bacterial carrier three plasmids were used which are based on the commercially available plasmid pCMV.beta.. This plasmid contains the structural gene of .beta.-gal under the control of the human cytomegalovirus (CMV) immediate early promoter and includes a splice donor and two splice acceptor sites in between the promoter and the structural gene. For studies examining the efficiency of the immune response against pathogens the .beta.-gal gene was replaced by genes encoding two virulence factors of Listeria monocytogenes. A truncated gene encoding a non-hemolytic variant of listeriolysin (pCMVhly) from amino acid positions 26 to 482 and a truncated variant of the structural gene of the membrane protein (pCMVactA) encoding amino acid 31 613 were used. S. typhimurium aroA strain SL7207 was transformed with these three plasmids and groups of mice were orally immunized by feeding 10.sup.8 organisms to each mouse per immunization. This dose was found to be optimal (data not shown). The mice did not show any overt signs of illness using this immunization schedule.

Induction of a Strong T Cell Response by Immunization with Salmonellae Carrying Eukaryotic Expression Vectors

The working hypothesis of these experiments is that orally administered S. typhimurium aroA would result in uptake of the bacteria by macrophages and/or dendritic cells, with concomitant activation by the endotoxin of the bacteria. Following a few rounds of bacterial division the intracellular bacteria would die because of their inability to synthesize essential aromatic amino acids. During this process plasmids would be released and transferred into the cytosol and the nucleus of the infected cells. Eventually, the encoded genes will be expressed by host APC.

The first prediction of this hypothesis is the induction of a strong cytotoxic response of CD8 T cells, since antigen would be expressed in the cytosol, the cellular compartment responsible for MHC class I presentation. To this end, two kinds of experiments were performed. Mice were either infected orally once with recombinant Salmonellae and their cytotoxic T cell responses were followed for several weeks by testing their spleen cells directly ex vivo (data not shown) or after one restimulation in vitro. Alternatively, mice were orally immunized four times at two weeks intervals and the course of the cytotoxic response was examined. FIG. 1 demonstrates that a strong and specific CD8 T cell response can be elicited with orally administered Salmonella carrying eukaryotic expression plasmids. Mice immunized with the truncated gene of listeriolysin elicited only a response towards targets sensitized with the immunodominant peptide comprising AA91-99 of listeriolysin (LLO) and not against targets sensitized with soluble hen egg lysozyme (HEL) or a control peptide (FIG. 1A). Similarly, spleen cells from mice immunized with Salmonella carrying the ActA expression plasmid could only respond to ActA (FIG. 1D). To reveal the cytotoxic response against ActA, we exploited the pore-forming activity of listeriolysin. This activity of listeriolysin allows the introduction of soluble passenger proteins into the cytosol of target cells (Darji et al., 1995a; Darji et al., 1997). Stimulators and target cells were therefore sensitized with a mixture of soluble ActA and LLO. A specific response was observed only when the combination of ActA and LLO was used. No response was found when LLO alone was tested. These responses were specific for the plasmid encoded antigen during the whole time period indicated in FIG. 1 panels B & C and E & F and were also observed when the response of mice immunized with Salmonella harboring the .beta.-gal control plasmid was studied (data not shown).

The kinetic of the responses indicated that even a single dose elicited a strong cytotoxic T cell response which peaked 5 weeks after immunization and then slowly declined (FIGS. 1C and F). On the other hand, the response was still rising even at the end of the observation period, i.e. 5 weeks after the last challenge in mice that had received four immunizing doses (FIGS. 1B and E). Thus, a strong cytotoxic response was observed when using Salmonella as potential vehicle for genetic immunization.

Genetic immunization usually also evokes a CD4 helper T cell response (Donnelly et al., 1997). Therefore, cells from spleen and mesenteric lymph nodes of the same mice used above were tested for their proliferative response against soluble proteins. This type of response is mainly due to presentation of antigen via MHC class II molecules and carried out by CD4 T cells. As shown in FIG. 2, a strong and specific helper T cell response, in parallel to the cytotoxic response is observed when eukaryotic expression plasmids carried by Salmonellae were used for immunization (FIGS. 2A and D). As with the CD8 response, a single dose was sufficient for a good response which was still increasing at the end of the observation period regardless of whether listeriolysin or ActA was used as antigen (FIGS 2C and F). Four consecutive immunizations however, resulted in an even stronger response which appeared long lasting since the response apparently was still increasing five weeks after the last challenge (FIGS. 2B and E). Similar results were obtained with Salmonella carrying the control plasmid expressing .beta.-gal (data not shown). Analysis of the supernatants of the in vitro cultures revealed production of IFN.gamma. by these T cells. No IL-4 could be found, suggesting that such an immunization scheme is mainly inducing a TH1 or inflammatory type of T helper response.

Induction of Specific Antibodies by Immunization with Salmonellae Carrying Eukaryotic Expression Vectors

Pooled sera of the groups of mice used above were tested for the presence of specific antibodies. Clearly, in addition to a cytotoxic and helper T cell response, immunization with Salmonellae carrying eukaryotic expression plasmids induced strong and specific antibody responses as revealed by ELISA (FIGS. 3A and B) or immunoblot (data not shown). Again a single immunization was sufficient for a good response which peaked four weeks after the administration of the bacteria and then declined in the same way as seen for cytotoxic response (FIGS. 3A and B). Four immunizations did not increase the antibody titer significantly but probably induced a longer lasting response since a plateau of antibody titer was not reached even at the end of the observation period (FIGS. 3A and B).

The analysis of the subclass distribution of individual mice at week 11 indicated a high concentration of IgG2a while the concentration of IgG2b and IgG3 was negligible (FIGS. 3C and D). This is in agreement with the finding that only IFN.gamma. and no IL-4 could be detected in the supernatant of the restimulated T helper cells. However, IgG1 was also observed at high concentrations in the immune sera. This subclass is found when TH2 helper cells are taking part in the immune response, indicating that under our experimental conditions TH2 cells might also be induced but were not revealed in the in vitro T cell assay. In addition, IgA antibodies were evoked by this immunization schedule (not shown).

Taken together the results presented in FIG. 1 3 show that immunization with S. typhimurium aroA carrying eukaryotic expression vectors can evoke responses in all three specific effector compartments of the immune system, namely, cytotoxic CD8 T cell, CD4 T cells and antibodies. The response in the T helper compartment was strongly biased towards a TH1 or inflammatory T helper response.

Protection Against Lethal Doses of L. monocytogenes

The strong response observed, in particular that of cytotoxic T cells, suggested that mice immunized in such a way should be protected from a lethal dose of L monocytogenes. Therefore, 90 days after the first immunization or 48 days after the fourth immunization--where applicable--mice were challenged i. v. with a dose of bacteria corresponding to 10.times.LD.sub.50. FIG. 4 shows that animals which were immunized four times consecutively with Salmonella e harboring an eukaryotic expression vector that encodes truncated LLO were completely protected (FIG. 4A). Animals that had received a single vaccination only were partially but significantly protected since at the time of termination of that experiment 60% of the animals were still alive. All animals that were immunized with Salmonellae that carried the .beta.-gal control plasmid were not protected and died within four days. Surprisingly, immunizations with Salmonellae carrying the ActA expression plasmid did not result in protection, although strong cytotoxic and helper T cell responses could be demonstrated in mice from the same group indicating that the immunization had been successfull (data not shown). Thus, the listerial membrane protein ActA is not a protective antigen.

Evidence for Transfer of the Expression Plasmid from the Carrier Salmonellae to Host Cells in vivo

We were concerned that a weak activity of the eukaryotic promoter in the bacteria or a cryptic prokaryotic promoter in the plasmid could result in expression of the antigens in the bacterial carrier thus eliciting the potent immune response. In fact, the recombinant Salmonellae harboring the pCMV.beta. exhibited low .beta.-gal activity (2.5 U) compared to the parental strain. To rule out any possibility, we immunized mice with a recombinant Salmonella strain that produced more than 100 fold higher levels (334 U) of .beta.-gal enzymatic activity. A single vaccinating dose using these bacteria did not elicit any measurable T cell or antibody response (FIG. 5A C). Repeated vaccination, however, resulted in a weak cytotoxic T cell response detectable after in vitro restimulation, although, it barely reached the strength of the response observed using a single immunization with Salmonellae harboring the eukaryotic expression plasmid of .beta.-gal (FIG. 5A). Neither a CD4 T cell nor an antibody response was observed even after repeated oral immunization with Salmonellae constitutively expressing .beta.-gal (FIGS. 5B and C).

As a result of the aroA mutation bacteria appear to die very quickly since live bacteria could never be demonstrated after immunization at various time points examined. Nevertheless, even at five weeks following oral administration of Salmonellae harboring the eukaryotic .beta.-gal expression plasmid, enzymatic activity of .beta.-gal could be detected in adherent cells--most likely macrophages--from the spleen of these mice suggesting plasmid transfer to the eucaryotic cell (data not shown). To further corroborate this observation we injected Salmonellae carrying the pCMV.beta. vector into the peritoneum of mice and harvested the peritoneal exudate cells after 1 hour. Cells were then cultured overnight in the presence of tetracycline to inhibit bacterial protein synthesis and finally stained for .beta.-gal activity. Enzymatic activity of .beta.-gal was observed in a large number of macrophage like cells. The staining was diffuse and clearly not restricted to the endocytic vesicles in which Salmonella usually reside. This suggests that plasmid DNA was transferred from dying Salmonellae to host cells and had occurred at a high frequency.

DNA Transfer from S. typhimurium aroA to Mammalian Host Cells in vitro

To obtain direct evidence that DNA transfer from the bacterial carrier to the mouse macrophages can take place, primary peritoneal macrophages were infected with Salmonellae harboring the .beta.-gal expression plasmid (pCMV.beta.). After infection for one hour, gentamicin was added to kill remaining extracellular bacteria. Four hours later tetracycline was added to kill resident intracellular bacteria. After overnight incubation, cells were stained for .beta.-gal activity. In up to 30% of the adherent, macrophage-like cells, enzymatic activity could be demonstrated even in the continuous presence of tetracycline which blocks bacterial protein synthesis (FIG. 6).

To show that .beta.-galactosidase was produced by the host cell, and not by the bacteria, two type of experiments were performed. Firstly, adherent peritoneal cells were infected and treated as described above. After overnight incubation RNA was extracted. If the plasmid had indeed been transferred and transcribed in the nucleus of the host cell, RNA splice products derived from the splice donor and acceptor sites within the vector should be demonstratable. By RT-PCR with a primer pair that hybridises to sequences on either side of the small intron, a PCR product could be observed which corresponded to one of the expected splice products (FIG. 7A). The identity of this product was confirmed by DNA sequencing (data not shown).

Secondly, biosynthetic labelling of proteins in the presence of tetracycline should only allow translation of mRNA produced by the eukaryotic host cells. Adherent peritoneal cells were infected as described and were pulsed for 30 min with .sup.35S-methionine after 4, 24 or 48 hours in the absence or presence of tetracycline. At four hours no .beta.-gal could be observed by immunoprecipitation, even in the absence of tetracycline where bacterial products should have been labelled (FIG. 7B). Thus, transfer of plasmid DNA and eukaryotic expression had not taken place yet. However, .beta.-gal could be immunoprecipitated following a 24 hour or 48 hour incubation period even when tetracycline was continuously present during both the incubation and labelling period. Preincubation of the anti-.beta.-gal antibody with an excess of unlabeled .beta.-gal demonstrated the specificity of the immunoprecipitation (FIG. 7B/lane 10). This clearly indicates that the .beta.-gal precipitated was produced by the infected mammalian host cell itself and not by the bacterium which had originally carried the expression plasmid. Thus, a transfer of the plasmid from Salmonellae to the host cell must have taken place.

Discussion

The transfer of eukaryotic expression plasmids from attenuated enteric bacteria into the nucleus of host cells has recently been demonstrated. While this work was in progress it was reported that auxotropic mutants of Shigella and E. coli that express the invasin of Shigella can carry eukaryotic expression plasmids into host cells (Sizemore et al., 1995; Courvalin et al., 1995). Given that both bacteria are capable of escape from the phagolysosome into the cytosol of the host cell, it follows that lysis of bacteria in this compartment would allow transfer of the released plasmid into the nucleus. Transfer of plasmid from intracellular pathogens such as Salmonella would be harder to imagine as these bacteria are generally retained within vacuoles of the infected host cell. Indeed, only a "low efficiency" of plasmid transfer into a macrophage cell line using attenuated Salmonella had been reported (Sizemore et al., 1995). Our initial experiments using several macrophage cell lines had also indicated that this was indeed the case (data not shown).

However, the kinetic and strength of the immune response after administering Salmonella carriing euckaryotic expression vectors suggested that a plasmid transfer might have taken place in vivo. We therefore decided to investigate primary macrophages isolated from the peritoneum of mice. Using these cells we could clearly demonstrate a transfer of an eucaryotic expression plasmid vector into host cells. A pathway that permits transfer of proteins from endocytic vesicles into the cytosol of some cell types including macrophages has been described (Reis de Sousa and Germain, 1995; Norbury et al., 1995). Whether such a pathway could also be responsible for the transfer of nucleic acids obseved here remains to be studied. The fact that plasmid transfer with Salmonella was only observed with primary macrophages and not with cell lines suggests the presence of a transport pathway which is only operating efficiently in primary cells.

Evidence for a transfer of plasmid DNA from Salmonella to the host cell in vitro is compelling. Splicing of RNA and protein synthesis in the presence of tetracycline are both only possible if the gene is expressed by the eukaryotic host cell. Evidence that transfer of the expression vector in vivo is responsible for induction of the strong immune response observed, also was obtained. Enzymatic activity of .beta.-gal could be observed five weeks after the last challenge in a few adherent spleen cells. However, viable Salmonella could not be detected even when tested one week after the last infection, thus, arguing that .beta.-gal expression cannot be due to residual surviving Salmonella. Nevertheless, it is intriguing how such antigen expressing cells can coexist in the presence of specific cytotoxic T cells.

Strong cytotoxic and, protective responses have only been reported with Salmonella that secrete the antigens. No comparable responses have been described using Salmonella that constitutively express nonsecreted heterologous proteins (Hess et al., 1996). High doses of recombinant bacteria that express intracellular protein were required to induce CD8 T cells (Turner et al., 1993). Although induction of specific antibodies have been described under some experimental conditions (Guzman et al., 1991; Walker et al., 1992) no antibody response was observed under the circumstances described above (Turner et al., 1993). This was confirmed by our own results (FIG. 5). We therefore find it highly unlikely that the strong responses of cytotoxic and helper T cells as well as the specific antibody production is the result of a fortuitous expression of the antigens in the Salmonella carrier.

The strength of the immune response observed especially after a single dose of immunization indicates that transfer of DNA by bacterial carrier is probably superior to a direct application of isolated plasmid DNA into skin or muscles. This suggests that by using the natural port of entry of a pathogen, the expression vector is transferred into cell types that have evolved to efficiently induce an immune response. It is likely that the Salmonella carrier is taken up by macrophages and dendritic cells. Whether, macrophages play a role during stimulation of naive T cells against bacteria is not clear, but dendritic cells are known to be highly efficient in priming resting T cells. Since the antigen is expressed in the cytosol of these cells a strong cytotoxic T cell response is to be expected.

The induction of an additionally strong helper and antibody response is puzzling and can only be speculated upon. Some cytosolic proteins can efficiently be presented by MHC class II molecules (Brooks and McCluskey 1993). However, it would be a very fortunate coincidence if all three proteins used in the present study display this property. In any case, it could not explain the antibody responses that we observed. It is more likely that APC expressing the antigen are lysed by specific cytotoxic cells and dying antigen containing cells or free antigen is taken up by neighbouring APC and presented via MHC class II molecules. The generated humoral response could be explained in a similar way.

In summary, oral genetic immunization using attenuated Salmonellae as carrier could work as schematically depicted in FIG. 8. Salmonella enter the host via M cells in the intestine. The bacteria are taken up in the dome areas by phagocytic cells such as macrophages and dendritic cells. These cells are activated by the pathogen and start to differentiate and probably to migrate into lymph nodes and spleen. During this time period the bacteria die due to their attenuating mutation and liberate the plasmid-based eukaryotic expression vectors. The plasmids are then transferred into the cytosol either via a specific transport system or by endosomal leackage. Finally, the vector enters the nucleus and is transcribed, thus, leading to antigen expression in the cytosol of the host cells. Specific cytotoxic T cells are induced by these activated APC which lyse antigen expressing cells. Free antigen or dying cells can be taken up by other APC, which now in turn can stimulate helper cells. Free antigen would also be responsible for the induction of an antibody response. In addition, bacterial endotoxin and DNA sequence motifs of the vector could also function as adjuvant and could contribute to the strength of the responses observed.

The helper T cell response induced with this type of genetic immunization seemed strongly biased to the TH1 type as indicated by IFNg production of restimulated T cells in vitro and the high titer of IgG2a in the humoral response (Mosmann and Coffman, 1989). This is not unexpected since bacteria usually induce inflammatory types of response. For many vaccination strategies it is desirable to induce an TH1 response for protection against the particular pathogen, e. g. strains of mice which respond with TH2 cells against Leishmania major do not clear the parasite and are not protected while mice which mount a TH1 response are resistant (Sher and Coffman, 1992). On the other hand, induction of TH2 type of responses or the conversion of a TH1 response into a TH2 response has been shown to be advantageous in inflammatory autoimmune diseases (Tian et al., 1996). Similarly, infections by nematodes might also require a TH2 response (Sher and Coffman, 1992). Since the bacteria are only being used as a vehicle in transferring the expression plasmids and therefore play only a secondary role it should be possible to manipulate the TH1 response. The induction of specific IgG1 suggests the presence of a TH2 component during the helper response that might be augmentable. Co-expression of the antigen together with certain cytokines or costimulatory molecules or alternatively using antisense strategies to suppress costimulatory molecules should make it possible to drive the responses more towards TH2.

Two well characterized virulence factors were tested as antigens for protection against a lethal challenge with L. monocytogenes. Listeriolysin has been shown before to induce protection (Harty and Bevan, 1992; Hess et al., 1996). This was also true under our experimental conditions. Interestingly, even a single dose of Salmonellae harboring the eukaryotic listeriolysin expression plasmid was sufficient to afford protection to 60% of the mice. On the other hand, ActA did not serve as protective antigen. The membrane protein ActA obviously is not available to the presentation mechanisms as long as the bacteria are alive. This raises the question as to whether membrane proteins of bacteria in general are not protective or whether ActA is a special case. Extensive phosphorylation of the ActA protein by host kinases following infection may affect its ability to be processed. Nevertheless, the role of bacterial surface-bound proteins in protection can now easily be addressed using the Salmonellae system for genetic vaccination.

The induction of a strong and specific antibody response which can be measured in ELISA and by immunoblot revealed additional benefits derived from the type of immunization described here. Thus, to raise specific polyclonal and possibly also monoclonal antibodies, any open reading frame can be inserted into an expression plasmid and used for immunization. This will facilitate the characterization of gene products where only sequence information is available.

In conclusion: using attenuated Salmonella which carry eukaryotic expression vectors, genetic immunization can be achieved by oral administration of the carrier. The stimulation of cytotoxic and helper T cells as well as the induction of a strong antibody response provides a very versatile system for new immunization strategies. The strength of this approach also draws on the development of newer more rationally attenuated Salmonellae strains as well as technical advances in providing conditional and targeted eukaryotic expression by the infected host cell. The possibility of genetic immunization with DNA fragments containing open reading frames will allow to define the function of new gene products, provide novel serological reagents, and permit delineation and assess efficacies of protective antigens in vaccination protocols.
 

Claim 1 of 15 Claims

1. An attenuated Salmonella strain comprising a eukaryotic expression vector, wherein said vector comprises a eukaryotic promoter and a heterologous DNA encoding a heterologous polypeptide, wherein said DNA is under the control of said eukaryotic promoter, wherein the attenuation is suitable for administration to a vertebrate, and wherein said administration to said vertebrate of said attenuated Salmonella strain results in expression of said polypeptide by said vertebrate and generates an immune response by said vertebrate to said polypeptide.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.