CA2288602A1 - Use of a secretion vector for fertility control by oral vaccination - Google Patents
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- A61P15/16—Masculine contraceptives
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- A—HUMAN NECESSITIES
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- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
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Abstract
The invention relates to a fertility-control method by oral vaccination using attenuated salmonellae or other gram-negative attenuated vaccination strains. Different systems of expression are used which enable a specific MHCII/CD4 or MHCI/CD8 immune response to be generated.
Description
Use of a Secretion Vector for Birth Control by Oral Vaccination The invention relates to a process for birth control by oral vaccination using attenuated salmonella or other gram-negative attenuated inoculation strains with use of various expression systems, which allow the production of a specific MHCII/CD4 or MHCI/CD8 immunological response.
Prior Art It has been known for a long time that women and men with significantly high antibody titers against human sperm are often infertile or have reduced fertility (Ingerslev and Ingerslev, 1989; Chen and Jones, 1981; Menge et al., 1982; Bronson et al., 1984). It was also shown that the immunization of female and male animals with extracts of whole sperm can result in inducing infertility (Kummerfeld and Foote, 1979; Munoz and Metz; 1978;
Tung et al., 1979; Menge et al., 1979). Primakoff et al. (1988a) used a monoclonal antibody to isolate the guinea pig-specific sperm-surface-antigen PH-20 and were able to show that the injection of this purified antigen in female or male guinea pigs produced a long-lasting immunization against fertility (1988b).
For a.number of other monoclonal antibodies, which are directed against ejaculated human sperm or isolated sperm of other species, it was possible to show that they cross-react with human sperm. Some react with components of seminal plasma and others detect antigens of testicular origin. Monoclonal antibodies, which immobilize or agglutinate human sperm or inhibit sperm binding and the penetration of zone-free hamsteroocytes, have been described. At present, several human sperm antigens are known, such as, for example, the M~ 95,000 antigen (Moore et al., 1987); the 55 kDa antigen, which is made obvious by the S36-37 mAbs (HSA-63) of Lee (Liu et al., 1990); and the human homologue of the sperm receptors of ZP-C (determined in mice by Saling and Bliel and Wassarman (Bliel, 1990; Leyton and Saling, 1989). The FA-1 antigen of mice and humans, which was partially identified (Naz, 1988) and the 24kDA antigen from the rat testis and human testis (Shaha et al., 1990) are of further interest. The antigen identified by Moore and Lee as well as the SP-10 immunogen were described as primary vaccine candidates (Anderson et al., 1987) by the "World Health Organization" (special area for birth-control vaccines). The three protein components of zona pellucida (ZP), an extracellular matrix that encases the nipples, are also suitable as antigens. These proteins are generally referred to as ZPA, ZPB and ZPC (Wassarmann, 1987, Science 235, 553-560). In this connection, the egg-sperm interaction is to be blocked or influenced by an immunological blocking. Tests with recombinantly produced ZPC, the zona protein, which is made responsible for the initial binding of the sperm to the zona, have also confirmed this. In all cases reported to date, however, irreversible damage of the ovary resulted in the case of long-term immunization (Skinner et al., 1984, Endocrinology 115, 2418-2432) . The mechanism of this loss of ovarian function is not explained up until now.
Prior Art It has been known for a long time that women and men with significantly high antibody titers against human sperm are often infertile or have reduced fertility (Ingerslev and Ingerslev, 1989; Chen and Jones, 1981; Menge et al., 1982; Bronson et al., 1984). It was also shown that the immunization of female and male animals with extracts of whole sperm can result in inducing infertility (Kummerfeld and Foote, 1979; Munoz and Metz; 1978;
Tung et al., 1979; Menge et al., 1979). Primakoff et al. (1988a) used a monoclonal antibody to isolate the guinea pig-specific sperm-surface-antigen PH-20 and were able to show that the injection of this purified antigen in female or male guinea pigs produced a long-lasting immunization against fertility (1988b).
For a.number of other monoclonal antibodies, which are directed against ejaculated human sperm or isolated sperm of other species, it was possible to show that they cross-react with human sperm. Some react with components of seminal plasma and others detect antigens of testicular origin. Monoclonal antibodies, which immobilize or agglutinate human sperm or inhibit sperm binding and the penetration of zone-free hamsteroocytes, have been described. At present, several human sperm antigens are known, such as, for example, the M~ 95,000 antigen (Moore et al., 1987); the 55 kDa antigen, which is made obvious by the S36-37 mAbs (HSA-63) of Lee (Liu et al., 1990); and the human homologue of the sperm receptors of ZP-C (determined in mice by Saling and Bliel and Wassarman (Bliel, 1990; Leyton and Saling, 1989). The FA-1 antigen of mice and humans, which was partially identified (Naz, 1988) and the 24kDA antigen from the rat testis and human testis (Shaha et al., 1990) are of further interest. The antigen identified by Moore and Lee as well as the SP-10 immunogen were described as primary vaccine candidates (Anderson et al., 1987) by the "World Health Organization" (special area for birth-control vaccines). The three protein components of zona pellucida (ZP), an extracellular matrix that encases the nipples, are also suitable as antigens. These proteins are generally referred to as ZPA, ZPB and ZPC (Wassarmann, 1987, Science 235, 553-560). In this connection, the egg-sperm interaction is to be blocked or influenced by an immunological blocking. Tests with recombinantly produced ZPC, the zona protein, which is made responsible for the initial binding of the sperm to the zona, have also confirmed this. In all cases reported to date, however, irreversible damage of the ovary resulted in the case of long-term immunization (Skinner et al., 1984, Endocrinology 115, 2418-2432) . The mechanism of this loss of ovarian function is not explained up until now.
It has now been found that by the insertion of genes or gene fragments that are suitable for birth control in a secretion vector that (a) contains the entire hemolysin operon including the hly-specific promoter and an enhancer-similar regulator hlyR and in which (b) a major part of the hlyA gene was deleted, (c) the proteins or protein fragments that are suitable for birth control are synthesized and (d) by the secretion of these antigens by attenuated salmonella or other gram-negative attenuated inoculation strains, (e) an oral vaccination is produced.
In a preferred embodiment of the invention, the genes or gene fragments that are suitable for birth control code for zona pellucida proteins.
In a preferred embodiment of the invention, the genes or gene fragments that are suitable for birth control code for zona pellucida proteins.
In another preferred embodiment, the secretion vector is pMOhlyl.
In another preferred embodiment, the salmonella strain is Salmonel~.a typhimurium.
In an especially preferred embodiment, the genes or gene fragments that are suitable for birth control code for zona pellucida proteins, the secretion vector is pMOhlyl and the salmonella strain is Salmonella typhimurium.
In a preferred embodiment of the invention, the genes or gene fragments that are suitable for birth control code for zona pellucida proteins.
In a preferred embodiment of the invention, the genes or gene fragments that are suitable for birth control code for zona pellucida proteins.
In another preferred embodiment, the secretion vector is pMOhlyl.
In another preferred embodiment, the salmonella strain is Salmonel~.a typhimurium.
In an especially preferred embodiment, the genes or gene fragments that are suitable for birth control code for zona pellucida proteins, the secretion vector is pMOhlyl and the salmonella strain is Salmonella typhimurium.
Genes or gene fragments that are suitable for birth control are defined as all genes or gene fragments that code for proteins or protein fragments that are suitable for birth control.
Most proteins, which are transported into the periplasma via the inside membrane of gram-negative bacteria; have an amino-terminal signal peptide, which is cleaved during this sec-dependent transport process (Pugsley, 1993). In contrast to this, the hemolysin (HlyA) of Escherichia coli is secreted into the extracellular medium via the inside and outside membrane with the aid of a secretion system. In contrast to the standard N-terminal transport signal of the sec-dependent protein transport, HlyA carries on the C-terminus a transport signal (HlyAs) that is formed by 50-60 amino acids (Hess et al., 1990; Jarchau et al., 1994). This HlyA signal is not cleaved during the course of secretion and itself has hardly any antigenic potential.
The hemolysin secretion system of E. coli is formed by three membrane proteins. Two of these proteins, HlyB (Gentschev &
Goebel, 1990) and HlyD (Sch~lein et al., 1992) sit in the inside membrane and are coded by genes that are part of the hemolysin determinant. The latter consists of an operon including the four genes hlyC, hlyA, hly8 and hlyD (Wagner et al., 1983; Hess et al., 1986) . The third protein of the translocation system, TolC, is located in the outside membrane (Wandersman & Delepelaire, 1990). The translocation of HlyA via both membranes of gram-negative bacteria requires energy in the form of ATP and a membrane potential of the inside membrane (Koronakis et al., 1995). In various cases, it was already shown that a fusion of HlyAS with other proteins or protein fragments is carried out in a secretion of these fusion proteins with the aid of the hemolysin secretion system (Blight & Holland 1994; Gentschev et al., 1994). In this case, the secretion efficiency depends on the folding and conformation of the reporter protein. The hemolysin secretion system of E. coli is functional in attenuated salmonella, which act as inoculation strains, and can be used to export fusion proteins (Gentschev et al., 1992; Su et al., 1992).
The genetic systems that are used in this connection were previously~based on two components: a plasmid, which carries genes (hly8 and hlyD) that are necessary for transport, and a plasmid, which allows the expression of fusion proteins (Gentschev et al., 1992; Hess et al., 1990).
The secretion vector pMOhlyl (Gentschev et al., 1995) carries the complete hemolysin operon (Goebel & Hedgpeth, 1982) including the hly-specific promoter and an "enhancer"-similar regulator hlyR (Vogel et al., 1988). A major part of the hlyA
gene was deleted, so that only 34 amino-terminal and 61 carboxy-terminal amino acids (HlyAs) were coded by HlyA. A singular Nsi I interface between an amino-terminal radical and a carboxy-terminal radical of HlyA allows an insertion of heterologous genes or gene fragments into the reading grid for HlyAs. The genetic information for antigens of a size of 10-1000 amino acids can be inserted into this secretion vector pMOhlyi, which makes possible the secretion of these antigens in attenuated salmonella and other gram-negative attenuated inoculation strains (e.g., E.
cola, vibrio aholerae, Yeresina enteroaolitica). Thus, in contrast to other secretion systems, the transport of heterologous fusion proteins by a single plasmid is made possible. In comparison to transport systems that were previously used for antigen presentation and that are suitable only for the transport of, in most cases, only relatively short peptides on the outside of the bacteria cell (Cardenas &
Clements, 1992), another important advantage of the hemolysin secretion apparatus is the considerably more variable size of the transport-competent proteins.
By manipulation of the HlyA secretion system, the same antigen can be presented in suitable attenuated gram-negative inoculation strains in cytoplasmatic form, surface-bound form or in secreted form. By using listeriolysin (from Listeria monooytogenes) that is made secretable in salmonella, it can additionally be achieved that the same antigen is processed in phagosome or enhanced in the cytosol of the antigen-presenting macrophage cell after infection with the antigen-producing salmonella-inoculation strain (Gentschev et al., 1995). With this possibility, it can be achieved that enhanced CD4+ or CD8+
T-cell responses are induced (Hess et al., 1996). Since, with this vector system, it was also possible to secrete cytokines and soluble cytokine receptors separately or in combination with the antigen to be produced in attenuated salmonella (Schiilein, 1993;
Gentschev, unpublished), in addition the immunological response can vary ( a . g . , as regards THE or TH2 ) .
The efficient secretion of proteins or protein fragments/HlyAS fusions that are suitable for birth control and r the high stability of the, vector make possible a completely novel, highly specific contraceptive vaccination with the aid of attenuated salmonella or other gram-negative attenuated inoculation strains.
After oral administration of the recombinant salmonella, the antigen (proteins or protein fragments that are suitable for birth control) is accessible to the immune system of the host by secretion of the proteins or protein fragment/HlyAs fusions that are suitable for birth control and depending on the protein epitope and according to the secretion path results in activation of B- and/or T-cells. Since HlyAs represents a weak antigen for B- and T-cells, antibodies and T-cells are induced by HlyAs-fusions and are mainly directed against the part of the reporter-antigen. Attenuated Salmonella and Yersinia strains, which secrete antigens, are suitable especially~for the induction of a humoral, mucosal immunological response and are therefore preferred. The induction of a mucosal immunity also results in the production of antigen-specific antibodies on remote mucosal surfaces. A B-cell response at the infection site also therefore results in stimulating a mucosal immunity in the reproductive tract.
With the invention that is disclosed here, it is possible the first time to produce directly in situ recombinant proteins or parts thereof without expensive production processes for the production of a corresponding immunological response, which results in contraception. With the aid of corresponding vectors, these proteins or protein sections in phagosomes can be brought to expression intracellularly or extracellularly. Humoral or cytotoxic immunological responses can thus be produced deliberately.
Description of the Figures Figure 1 shows a vector map of the secretion plasmid pMOhlyi.
Figure 2 shows a Western-blot, in which various pMOhlyl/(hu)ZPA/constructs were tested for expression and secretion. After transformation of E. coli DHSa with the various pMOhlyl/(hu)ZPA/constructs, 2 ml of supernatant from overnight cultures was precipitated with 10% (v/v) TCA (trichloroacetic acid) for 4 hours on ice, pelletized by centrifuging and resuspended in SDS-sample buffer. The supernatant proteins were separated by SDS-PAGE (polyacrylamide gel electrophoresis) in a 15% polyacrylamide gel and tested for expression and secretion after transfer to nitrocellulose with a polyclonal antibody, which is directed against the hemolysin portion of the fusion protein. A1 stands for the huZPA-1 construct (see Figure 7); A4 stands for the huZPA-4 construct (see Figure 7), pM0 stands for the vector control and B4 stands for the huZPB-4 construct (see Figure 8).
Figure 3 shows a Western-blot, in which various pMOhlyl/(hu) ZPB/constructs were tested for expression and secretion. After transformation of E. coli DHSa with the various pMOhly1/(hu)ZPB
constructs, 2 ml of supernatant from overnight cultures were precipitated with 10% (v/v) TCA (trichloroacetic acid) for 4 hours on ice, pelletized by centrifuging and resuspended in SDS-sample buffer. The supernatant proteins were separated by SDS-PAGE (polyacrylamide gel electrophoresis) in a 15% polyacrylamide gel and tested for expression and secretion after transfer to nitrocellulose with a polyclonal antibody, which is directed against the hemolysin portion of the fusion protein. B1 stands for the huZPB-1 construct (see Figure 8); B2 stands for the huZPB-2 construct (see Figure 8); B3 stands for the huZPB-3 construct (see Figure 8); B4 stands for the huZPB-4 construct (see Figure 8); B5 stands of the huZPB-5 construct (see Figure 8) and pM0 stands for the vector control.
Figure- 4 shows a Western-blot, in which various pMOhlyi/(hu) ZPC constructs were tested for expression and secretion. After transformation of E. coli DHSa with the various pMOhlyi/(hu)ZPC
constructs, 2 ml of supernatant from overnight cultures was precipitated with 10% (v/v) TCA (trichloroacetic acid) for 4 hours on ice, pelletized by centrifuging and resuspended in SDS-sample buffer. The supernatant proteins were separated by SDS-PAGE (polyacrylamide gel electrophoresis) in a 15% polyacrylamide gel and tested for expression and secretion after transfer to nitrocellulose with a polyclonal antibody, which is directed against the hemolysin portion of the fusion protein. C1 stands for the huZPC-1 construct (see Figure 9); C2 stands for the huZPC-2 construct (see Figure 9); C3 stands for the huZPC-3 construct (see Figure 9) and pM0 stands for vector control.
Figure 5 shows a Western-blot, in which various pMOhlyi/(m) ZPB/constructs were tested for expression and secretion. After transformation of E. coli DHSa with the various pMOhlyl/(m) ZPB/constructs, 2 ml of supernatant from overnight cultures were precipitated with 10% (v/v) TCA (trichloroacetic acid) for 4 hours on ice, pelletized by centrifuging and resuspended in SDS-sample buffer. The supernatant proteins were separated by SDS-PAGE (polyacrylamide gel electrophoresis) in a 15% polyacrylamide gel and tested for expression and secretion after transfer to nitrocellulose with a polyclonal antibody, which is directed against the hemolysin portion of the fusion protein. B1 stands for the mZPB-1 construct (see Figure 10); B2 stands for the mZPB-2 construct (see Figure 10); B3 stands for the mZPB-3 construct (see Figure 10); B4 stands for the mZPB-4 construct (see Figure 10), and pM0 stands for the vector control.
Figure 6 shows a Western-blot, in which various pMOhlyl (hu)ZPB/constructs were tested for expression and secretion.
After transformation of S, typhimurium SL7207 with the various pM0/(hu)ZPB/constructs, 2 ml of supernatant from overnight cultures was precipitated with 10% (v/v) TCA (trichloroacetic acid) for 4 hours on ice, pelletized by centrifuging and resuspended in SDS-sample buffer. The supernatant proteins were separated by SDS-PAGE (polyacrylamide gel electrophoresis) in a 15% polyacrylamide gel and tested for expression and secretion after transfer to nitrocellulose with a polyclonal antibody, which is directed against the hemolysin portion of the fusion protein. B1 stands for the huZPB-1 construct (see Figure 8); B2 stands for the huZPB-2 construct (see Figure 8); B3 stands for the huZPB-3 construct (see Figure 8); B4 stands for the huZPB-4 construct (see Figure 8); B5 stands for the huZPB-5 construct (see Figure 8) and pM0 stands for the vector control.
Figure 7 shows a Western-blot, in which various pMOhlyl(hu)ZPC/constructs were tested for expression and secretion. After transformation of Yersinia enterocolitiaa Wap Yv-515 with the various pM0/(hu)ZPC/constructs, 2 ml of supernatant from overnight cultures was precipitated with 10%
(v/v) TCA (trichloroacetic acid) for 4 hours on ice, pelletized by centrifuging and resuspended in SDS-sample buffer. The supernatant proteins were separated by SDS-PAGE (polyacrylamide gel electrophoresis) in a 15% polyacrylamide gel and tested for expression and secretion after transfer to nitrocellulose with a polyclonal antibody, which is directed against the hemolysin portion of the fusion protein. C1 stands for the huZPC-1 construct (see Figure 12); C2d stands for the huZPC-2 construct with a double insert (see Figure 12); C3d stands for the huZPC-3 construct with a double insert (see Figure 12); C4 stands for the huZPC-4 construct (see Figure 12), and.pMO stands for the vector control.
Figure 8 shows a gene map of the cDNA of huZPA. The arrows show the positions of the primer that is selected for a PCR
amplification (Table 1), whereby the numbers indicate the starting point on the 5'-end of the primer relative to the starting codon of the cDNA sequence of the gene. The solid rectangles show the amino-terminal transport signal, the putative fusion gap site and a strongly hydrophobic area on the C-terminus that could act as a membrane anchor. The fragments that are amplified by PCR are depicted in the lower part with starting and ending points, which are produced by an Nsi-I restriction endonuclease digestion of the PCR products and henceforth referred to as huZPA-1, huZPA-2, etc. In addition, the length of the fragments is indicated both in base pairs (bp) and in amino acids (aa). The calculated molecular weights are reproduced for the zona fragments and in parentheses for the zona/HlyA fusion proteins in kDA. Finally, it is indicated whether a secretion of the fusion proteins with an antibody that is directed against the HlyA portion was detected.
Figure 9 shows a gene map of the CDNA of huZPB. The arrows show the positions of the primer that is selected for a PCR
amplification (Table 1), whereby the numbers indicate the starting point on the 5'-end of the primer relative to the starting codon of the cDNA sequence of the gene. The solid rectangles show the amino-terminal transport signal, the putative fusion gap site and a strongly hydrophobic area on the C-terminus that could act as a membrane anchor. The fragments that are amplified by PCR are depicted in the lower part with starting and ending points, which are produced by an Nsi-I restriction endonuclease digestion of the PCR products and henceforth referred to as huZPB-1, huZPB-2. In addition, the length of the fragments is indicated both in base pairs (bp) and in amino acids (aa). The calculated molecular weights are reproduced for the zona fragments and in parentheses for the zona/HlyA fusion proteins in kDa. Finally, it is indicated whether a secretion of the fusion proteins with an antibody that is directed against the HlyA portion was detected.
Figure 10 shows a gene map of cDNA of huZPC. The arrows show the positions of the primer that is selected for a PCR
amplification (Table 1), whereby the numbers indicate the starting point on the 5'-end of the primer relative to the starting codon of the cDNA sequence of the gene. The solid rectangles show the amino-terminal transport signal, the putative fusion gap site and a strongly hydrophobic area on the C-terminus that could act as a membrane anchor. The fragments that are amplified by PCR are depicted in the lower part with starting and ending points, which are produced by an Nsi-I restriction endonuclease digestion of the PCR products and henceforth referred to as huZPC-1, huZPC-2, etc. In addition, the length of the fragments is indicated both in base pairs (bp) and in amino acids (aa). The calculated molecular weights are reproduced for the zona fragments and in parentheses for the zona/HlyA fusion proteins in kDa. Finally, it is indicated whether a secretion of the fusion proteins with an antibody that is directed against the HlyA portion was detected.
Figure 11 shows a gene map of the cDNA of mZPB. The arrows show the positions of the primer that is selected for a PCR
amplification (Table 1), whereby the numbers indicate the starting point on the 5'-end of the primer relative to the starting codon of the cDNA sequence of the gene. The solid rectangles show the amino-terminal transport signal, the putative fusion gap site and a strongly hydrophobic area on the C-terminus that could act as a membrane anchor. The fragments that are amplified by PCR are depicted in the lower part with starting and ending points, which are produced by an Nsi-I restriction endonuclease digestion of the PCR products and henceforth referred to as mZPB-1, huZPB-2, etc. In addition, the length of the fragments is indicated both in base pairs (bp) and in amino acids (aa). The calculated molecular weights are reproduced for the zona fragments and in parentheses for the zona/HlyA fusion proteins in D. Finally, it is Figure 12 shows a gene map of cDNA of huZPC. The arrows show the positions of the primer that is selected for a PCR
amplification (Table 1), whereby the numbers indicate the starting point on the 5~-end of the primer relative to the starting codon of the cDNA sequence of the gene. The fragments that are amplified by PCR are depicted in the lower part with starting and ending points, which are produced by Nsi-I
restriction endonuclease digestion of the PCR products and henceforth referred to as huZPC-1, huZPC-2, etc. In addition, the length of the fragments is indicated both in base pairs (bp) and in amino acids (aa). The calculated molecular weights are reproduced for the zona fragments and in parentheses for the zona/HlyA fusion proteins in kDa. Finally, it is indicated whether a secretion of the fusion proteins with an antibody that is directed against the HlyA portion was detected.
The assumption is that one skilled in the art can execute this invention to its full scope based on the description. Many known techniques and protocols for manipulating nucleic acids, such as, for example, metagenesis, sequencing, the introduction of nucleic acid in cells or the analysis of proteins is in detail in Short Protocols in Molecular Biology, Second Edition, Ausrubel et al. Eds., John Wiley & Sons, 1992; Molecular Cloning: A
Laboratory Manual: 2nd Edition, Sambrock~et al., 1989, Cold Spring Harbor Laboratory Press. The content of Sambrook et al.
and Ausrubel et al. is incorporated herewith as reference.
This invention contains pharmaceutical preparations for all uses and the methods that are described. The invention thus contains, for example, pharmaceutical preparations that contain the attenuated bacteria with the secretion vector according to this invention. The oral administration is carried out preferably in the form of encapsulated, biodegradable polymers, for example PLPG.
The following examples are used only for a more detailed description and show that the invention is feasible, and the examples are in no way to act in a limiting manner.
Example 1 Cloning and Expression of huZPA, huZPB, huZPC and mZPB Fragments in E. aoli and 8. typhimurium The plasmids pGEX-KG-huZPA, pGEX-KG-huZPB and pGEX-KG-huZPC
(Peter Bringmann, Schering AG), which carry the cDNA of the human ZPA, ZPB and ZPC gene and the ovarian mRNA of mice, which made possible the cDNA synthesis of the mouse ZPB-gene, are the starting point of the cloning strategy. Starting from mRNA and isolated from the ovaries of superovulating mice (Uwe Eberspacher, Schering AG), the corresponding CDNA was synthesized as follows: about 5 ~g of RNA was mixed in 32 ~C1 of DEPC-H20 with 3 ~,l of oligo-dT-primer and incubated for 5 minutes at 65°C.
The batch is allowed to cool for 10 minutes at room temperature, and the following reagents are added: 5 ~,1 of synthesis buffer, ~1 of 0.1 M DTT, 1 ~C1 of RNase inhibitor, 3 ul of 25 mmol of dNTPs and 1 ~,1 of MMLV reverse transcriptase (20 U/~C1) (1st Strand Synthesis Kit, Stratagene). An incubation is carried out for 1 hour at 37°C. With the aid of specific oligonucleotides (Table 1), gene fragments that overlap by PCR from the corresponding cDNA were amplified, which have 5' and 3' Nsi I-restriction interfaces (Figures 8 to 12: maps of the ZP-genes with indication of the primer positions and the gene fragments that result from the above). The PCR-amplification (Saiki et ~al., 1988) was performed in a Thermocycler 60/2 (bio-med, Theres, Germany). For this purpose, cDNA was amplified with the corresponding 5'- and 3'-primers (1 ~mol each of final concentration), dNTPs (200 ~,mol each) and 2.5 U Taq DNA-polymerase (Promega) and the buffer of the manufacturer in a volume of 100 ~,1. Before the first cycle, a denaturation took place for 3 minutes at 91°C. 30 cycles then followed under the following conditions: 1 minute of denaturation at 91°C, 1 minute of annealing at 55°C and 1 minute of extension at 72°C. The reaction products were electrophoretically separated in a 2%
agarose gel and made visible by coloration with ethidium bromide.
These PCR products were first inserted "blunt" into the Sma I-interface of the vector pUCl8. The ZP-fragment was then cut out by Nsi I-digestion and inserted into the expression vector pMOhlyi (Figure 1). After transformation of E. coli DHSa with the various pMOhlyl/ZP-constructs, 2 ml of supernatant from overnight cultures was precipitated with 10% (v/v) TCA for 4 hours on ice, pelletized by centrifuging and resuspended in the SDS-sample buffer. The supernatant proteins were separated by SDS-PAGE in a 15% polyacrylamide gel and tested for the secretion of zona/hemolysin-fusion proteins after transfer to nitrocellulose. With the aid of a polyclonal antibody, which is directed against the hemolysin portion of the fusion protein, the expression and secretion of two huZPA fragments (A1 and A4, Figure 2) and four huZPB-fragments (B1-B4, Figure 3) could be detected. For huZPC, a transport-competent domain was identified (C1, Figure 4). Analogously to huZPB, the secretion of three protein fragments from mZPB could also be shown by the transport system (B1-B3, Figure 5).
Then, the transformation of the described pMOhly1 derivatives took place in the 8. typhimurium LB5000 strain, in which the restriction system is absent, so that E. coli-DNA can be introduced efficiently in this strain. After the recombinant expression vectors were isolated from 8. typhimurium LB5000, it was possible to transform the 8. typhimurium SL7207 strain successfully. As already described several times, this strain is suitable as an inoculation strain. The secretion of the ZP/Hly-fusion proteins also is carried out in this strain. By way of example, Figure 6 shows the expression and the secretion of the huZPB fragments in salmonella. After oral administration in mice, these salmonella that express ZP/Hly should induce a mucosal immunity, which is also directed specifically against the ZP-portion of the fusion protein.
Example 2 Cloning and Expression of huZPC, huZPB, huZPC and mZPB Fragments in Y. enterocolitica The plasmids pGEX-KG-huZPA, pGEX-KG-huZPB and pGEX-KG-huZPC
(Peter Bringmann, Schering AG), which carry the cDNA of the human ZPA, ZPB and ZPC gene and the ovarian mRNA of mice, which made possible the cDNA synthesis of the mouse ZPB-gene, are the starting point of the cloning strategy. Starting from mRNA and isolated from the ovaries of superovulating mice (Uwe Ebersp~cher, Schering AG), the corresponding cDNA was synthesized as follows: about 5 ~Cg of RNA was mixed in 32 ~C1 of DEPC-H20 with 3 ~C1 of oligo-dT-primer and incubated for 5 minutes at 65°C.
The batch is allowed to cool for 10 minutes at room temperature, and the following reagents are added: 5 ~,1 of synthesis buffer, 5 ul of 0.1 M DTT, 1 ~1 of RNase inhibitor, 3 ~1 of 25 mmol of dNTPs and 1 ~1 of MMLV reverse transcriptase (20 U/~Cl) (1st Strand Synthesis Kit, Stratagene). An incubation is carried out for 1 hour at 37°C. With the aid of specific oligonucleotides (Table 1), gene fragments that overlap by PCR from the corresponding cDNA were amplified, which have 5' and 3' Nsi I-restriction interfaces (Figures 8 to 12: maps of the ZP-genes with indication of the primer positions and the gene fragments that result from the above). The PCR-amplification (Saiki et al., 1988) was performed in a Thermocycler 60/2 (bio-mad, Theres, Germany). For this purpose, cDNA was amplified with the corresponding 5'- and 3'-primers (1 ~mol each of final concentration), dNTPs (200 ~mol each) and 2.5 U Taq DNA-polymerase (Promega) and the buffer of the manufacturer in a volume of 100 ~1. Before the first cycle, a denaturation took place for 3 minutes at 91°C. 30 cycles then followed under the following conditions: 1 minute of denaturation at 91°C, 1 minute of annealing at 55°C and 1 minute of extension at 72°C. The reaction products were electrophoretically separated in a 2%
agarose gel and made visible by coloration with ethidium bromide.
These PCR products were first inserted "blunt" in the Sm I-interface of the vector pUCl8. The ZP-fragment was then cut out by Nsi I-digestion and -inserted in the expression vector pMOhlyi (Figure 1). After transformation of E. coli DHSa with the various pMOhlyl/ZP-constructs, 2 ml of supernatant from overnight cultures was precipitated with 10% (v/v) TCA for 4 hours on ice, palletized by centrifuging and resuspended in the SDS-sample buffer. The supernatant proteins were separated by SDS-PAGE in a 15% polyacrylamide gel and tested for the secretion of zona/hemolysin-fusion proteins after transfer to nitrocellulose.
With the aid of a polyclonal antibody, which is directed against the hemolysin portion of the fusion protein, the expression and secretion of huZPA fragments, huZPB-fragments and huZPC could be detected (see above). Then, the transformation of the described pMOhlyl derivatives took place in the Y. enteroaolitica LB5000 strain, in which the restriction system is absent, so that E.
cola-DNA can be introduced efficiently in this strain. After the recombinant expression vectors were isolated from Y.
enterocolitica LB5000, it was possible to transform the Y.
enterocolitica Wap Yv-515 strain successfully. As already described several times, this strain is suitable as an inoculation strain. The secretion of the ZP/Hly-fusion proteins also is carried out in this strain. By way of example, Figure 7 shows the expression and the secretion of the huZPC fragments in Y. enterocolitica. After oral administration in mice, these yersinia that express ZP/Hly should induce a mucosal immunity, which is also directed specifically against the ZP-portion of the fusion protein.
Table 1: Primer sequences for the amplification of ZP-gene fragments by PCR and expected PCR-product values [Key:] PCR-Produkt = PCR product ZP-Fragment PCR-Produkt (bp) huZPA
huZPA1 5'-GAAATGGCATGCATGCATCTGTG-3'390 5'-GCTGAAGCATGCATTCATGGCCTC-3' huZPA2 5'-CTGCCAGATGCATTGAAGGAAGGC-3'408 ~
5'-GTCAGCTTGAATGCATCTAAGTAGAAC-3' ~ huZPA3 5'-CGAAATTATATGCATAATGCCTAC-3'456 !
I
_ _ _ 5'-GAGTAAGGCATGCATCGTTGATG-3' huZPA4 5'-GTGAAGTATGCATATAGCAGG-3' 318 _ _ 5'-CATATGCACATGCATCCACGACAAC-3' huZPA5 5'-CACCATGGATGCATACTCTTTCC-3'249 5'-GCCTAGAGGATGCATGGCAGGTCAG3' huZPB
huzPB1 5'-GCTTCCAGTATGCATTAAACCTC-3'207 5'-CGCGCCTGATGCATCAACTCCAAC-3' huZPB2 5-GTGGTGTfGGATGCATCCTATAGC-3'399 5'-CAGGGTTACATGCATfGTCATTCC-3' huZPB3 5'-CTCTfGGATGCATTGCGCTTGGCC-3'375 5'-CTTCACCAATGCATAGTCACCAAC-3' huZPB4 5'-GAGACCCAGCATGCATCCCTCACTC-f498 5-GCCTTTGCTAGATGCATTAGCAGTAG-3' huZPBS 5'-GCCAGCCTGCTGATGCATCATCCTG-3'213 5'-CAGCCAAGTAGGATGCATACAAGGC-3' ZP-Fragment PCR-Produkt ~ (bp) huZPC
I huZPC1 5'-GCCAGCCATGCATAGACGTCCGTAC-3'495 ~ ~
- -5'-CAGTGTGGATTT_ATGC_ATGGAGGTG-3' i huZPC2 5'-GAGAACTGGAATGCATAGAAGAGG-3'366 ~
5'CCAGCTGTTGGATGCATTGCTGAAG-3' huZPC2' ~ 5-CAATGTGAGCAGCCATGCATTCCTGC-3'~ 285 ' - - i 5'-GAATGCAGAAGA_TGCATCAGTGAG-3' i huZPC3 ~ 5'-CAGGACCCAGATGCATTCAACAAG-3'~ 363 - -5'-GAGAC~C~G 1 C~GGA I GGP. r I Gi.:GAGAC:-3' ' i I
I h Z C3' S'-GACCAGAATGCATCCCCTTATCAG3'360 ~
5'-GGTTACGGGATGCATACCTGGAC-3', I
~
huZPC4 5'GCCAGCCATGCATATACGTCCGTAC-3'936 ~
5'-GGTTACGGGATGCATACCTGGAG3' mZPB
mZPB1 ~ 5'-GAATACAGCTATGCATGTGGGGTAC-3'450 ~
5'-GCAGGAGATGCATGGATGAAGCTC-3' mZP82 5'-CACACCTTGGATGCATCTGGCCAG3'408 5'-CATCCGTTGGATGCATAGGCCAG-3' mZPB3 5'-GCCTfGACACATG_CA_TTCCTGCTAG-3'339 5'-CTTATCCGATGCATTCCTCAGCTG3' mZPB4 5'-GTGACTCAATATGCATCCCTGAGGC-3'393 5'-CAGAGGGTGGCATGCATAGGCACTAC-3' Underlined bases indicate a deviation from the respective ZP-sequence to introduce the Nsi I interface that is necessary for cloning
Most proteins, which are transported into the periplasma via the inside membrane of gram-negative bacteria; have an amino-terminal signal peptide, which is cleaved during this sec-dependent transport process (Pugsley, 1993). In contrast to this, the hemolysin (HlyA) of Escherichia coli is secreted into the extracellular medium via the inside and outside membrane with the aid of a secretion system. In contrast to the standard N-terminal transport signal of the sec-dependent protein transport, HlyA carries on the C-terminus a transport signal (HlyAs) that is formed by 50-60 amino acids (Hess et al., 1990; Jarchau et al., 1994). This HlyA signal is not cleaved during the course of secretion and itself has hardly any antigenic potential.
The hemolysin secretion system of E. coli is formed by three membrane proteins. Two of these proteins, HlyB (Gentschev &
Goebel, 1990) and HlyD (Sch~lein et al., 1992) sit in the inside membrane and are coded by genes that are part of the hemolysin determinant. The latter consists of an operon including the four genes hlyC, hlyA, hly8 and hlyD (Wagner et al., 1983; Hess et al., 1986) . The third protein of the translocation system, TolC, is located in the outside membrane (Wandersman & Delepelaire, 1990). The translocation of HlyA via both membranes of gram-negative bacteria requires energy in the form of ATP and a membrane potential of the inside membrane (Koronakis et al., 1995). In various cases, it was already shown that a fusion of HlyAS with other proteins or protein fragments is carried out in a secretion of these fusion proteins with the aid of the hemolysin secretion system (Blight & Holland 1994; Gentschev et al., 1994). In this case, the secretion efficiency depends on the folding and conformation of the reporter protein. The hemolysin secretion system of E. coli is functional in attenuated salmonella, which act as inoculation strains, and can be used to export fusion proteins (Gentschev et al., 1992; Su et al., 1992).
The genetic systems that are used in this connection were previously~based on two components: a plasmid, which carries genes (hly8 and hlyD) that are necessary for transport, and a plasmid, which allows the expression of fusion proteins (Gentschev et al., 1992; Hess et al., 1990).
The secretion vector pMOhlyl (Gentschev et al., 1995) carries the complete hemolysin operon (Goebel & Hedgpeth, 1982) including the hly-specific promoter and an "enhancer"-similar regulator hlyR (Vogel et al., 1988). A major part of the hlyA
gene was deleted, so that only 34 amino-terminal and 61 carboxy-terminal amino acids (HlyAs) were coded by HlyA. A singular Nsi I interface between an amino-terminal radical and a carboxy-terminal radical of HlyA allows an insertion of heterologous genes or gene fragments into the reading grid for HlyAs. The genetic information for antigens of a size of 10-1000 amino acids can be inserted into this secretion vector pMOhlyi, which makes possible the secretion of these antigens in attenuated salmonella and other gram-negative attenuated inoculation strains (e.g., E.
cola, vibrio aholerae, Yeresina enteroaolitica). Thus, in contrast to other secretion systems, the transport of heterologous fusion proteins by a single plasmid is made possible. In comparison to transport systems that were previously used for antigen presentation and that are suitable only for the transport of, in most cases, only relatively short peptides on the outside of the bacteria cell (Cardenas &
Clements, 1992), another important advantage of the hemolysin secretion apparatus is the considerably more variable size of the transport-competent proteins.
By manipulation of the HlyA secretion system, the same antigen can be presented in suitable attenuated gram-negative inoculation strains in cytoplasmatic form, surface-bound form or in secreted form. By using listeriolysin (from Listeria monooytogenes) that is made secretable in salmonella, it can additionally be achieved that the same antigen is processed in phagosome or enhanced in the cytosol of the antigen-presenting macrophage cell after infection with the antigen-producing salmonella-inoculation strain (Gentschev et al., 1995). With this possibility, it can be achieved that enhanced CD4+ or CD8+
T-cell responses are induced (Hess et al., 1996). Since, with this vector system, it was also possible to secrete cytokines and soluble cytokine receptors separately or in combination with the antigen to be produced in attenuated salmonella (Schiilein, 1993;
Gentschev, unpublished), in addition the immunological response can vary ( a . g . , as regards THE or TH2 ) .
The efficient secretion of proteins or protein fragments/HlyAS fusions that are suitable for birth control and r the high stability of the, vector make possible a completely novel, highly specific contraceptive vaccination with the aid of attenuated salmonella or other gram-negative attenuated inoculation strains.
After oral administration of the recombinant salmonella, the antigen (proteins or protein fragments that are suitable for birth control) is accessible to the immune system of the host by secretion of the proteins or protein fragment/HlyAs fusions that are suitable for birth control and depending on the protein epitope and according to the secretion path results in activation of B- and/or T-cells. Since HlyAs represents a weak antigen for B- and T-cells, antibodies and T-cells are induced by HlyAs-fusions and are mainly directed against the part of the reporter-antigen. Attenuated Salmonella and Yersinia strains, which secrete antigens, are suitable especially~for the induction of a humoral, mucosal immunological response and are therefore preferred. The induction of a mucosal immunity also results in the production of antigen-specific antibodies on remote mucosal surfaces. A B-cell response at the infection site also therefore results in stimulating a mucosal immunity in the reproductive tract.
With the invention that is disclosed here, it is possible the first time to produce directly in situ recombinant proteins or parts thereof without expensive production processes for the production of a corresponding immunological response, which results in contraception. With the aid of corresponding vectors, these proteins or protein sections in phagosomes can be brought to expression intracellularly or extracellularly. Humoral or cytotoxic immunological responses can thus be produced deliberately.
Description of the Figures Figure 1 shows a vector map of the secretion plasmid pMOhlyi.
Figure 2 shows a Western-blot, in which various pMOhlyl/(hu)ZPA/constructs were tested for expression and secretion. After transformation of E. coli DHSa with the various pMOhlyl/(hu)ZPA/constructs, 2 ml of supernatant from overnight cultures was precipitated with 10% (v/v) TCA (trichloroacetic acid) for 4 hours on ice, pelletized by centrifuging and resuspended in SDS-sample buffer. The supernatant proteins were separated by SDS-PAGE (polyacrylamide gel electrophoresis) in a 15% polyacrylamide gel and tested for expression and secretion after transfer to nitrocellulose with a polyclonal antibody, which is directed against the hemolysin portion of the fusion protein. A1 stands for the huZPA-1 construct (see Figure 7); A4 stands for the huZPA-4 construct (see Figure 7), pM0 stands for the vector control and B4 stands for the huZPB-4 construct (see Figure 8).
Figure 3 shows a Western-blot, in which various pMOhlyl/(hu) ZPB/constructs were tested for expression and secretion. After transformation of E. coli DHSa with the various pMOhly1/(hu)ZPB
constructs, 2 ml of supernatant from overnight cultures were precipitated with 10% (v/v) TCA (trichloroacetic acid) for 4 hours on ice, pelletized by centrifuging and resuspended in SDS-sample buffer. The supernatant proteins were separated by SDS-PAGE (polyacrylamide gel electrophoresis) in a 15% polyacrylamide gel and tested for expression and secretion after transfer to nitrocellulose with a polyclonal antibody, which is directed against the hemolysin portion of the fusion protein. B1 stands for the huZPB-1 construct (see Figure 8); B2 stands for the huZPB-2 construct (see Figure 8); B3 stands for the huZPB-3 construct (see Figure 8); B4 stands for the huZPB-4 construct (see Figure 8); B5 stands of the huZPB-5 construct (see Figure 8) and pM0 stands for the vector control.
Figure- 4 shows a Western-blot, in which various pMOhlyi/(hu) ZPC constructs were tested for expression and secretion. After transformation of E. coli DHSa with the various pMOhlyi/(hu)ZPC
constructs, 2 ml of supernatant from overnight cultures was precipitated with 10% (v/v) TCA (trichloroacetic acid) for 4 hours on ice, pelletized by centrifuging and resuspended in SDS-sample buffer. The supernatant proteins were separated by SDS-PAGE (polyacrylamide gel electrophoresis) in a 15% polyacrylamide gel and tested for expression and secretion after transfer to nitrocellulose with a polyclonal antibody, which is directed against the hemolysin portion of the fusion protein. C1 stands for the huZPC-1 construct (see Figure 9); C2 stands for the huZPC-2 construct (see Figure 9); C3 stands for the huZPC-3 construct (see Figure 9) and pM0 stands for vector control.
Figure 5 shows a Western-blot, in which various pMOhlyi/(m) ZPB/constructs were tested for expression and secretion. After transformation of E. coli DHSa with the various pMOhlyl/(m) ZPB/constructs, 2 ml of supernatant from overnight cultures were precipitated with 10% (v/v) TCA (trichloroacetic acid) for 4 hours on ice, pelletized by centrifuging and resuspended in SDS-sample buffer. The supernatant proteins were separated by SDS-PAGE (polyacrylamide gel electrophoresis) in a 15% polyacrylamide gel and tested for expression and secretion after transfer to nitrocellulose with a polyclonal antibody, which is directed against the hemolysin portion of the fusion protein. B1 stands for the mZPB-1 construct (see Figure 10); B2 stands for the mZPB-2 construct (see Figure 10); B3 stands for the mZPB-3 construct (see Figure 10); B4 stands for the mZPB-4 construct (see Figure 10), and pM0 stands for the vector control.
Figure 6 shows a Western-blot, in which various pMOhlyl (hu)ZPB/constructs were tested for expression and secretion.
After transformation of S, typhimurium SL7207 with the various pM0/(hu)ZPB/constructs, 2 ml of supernatant from overnight cultures was precipitated with 10% (v/v) TCA (trichloroacetic acid) for 4 hours on ice, pelletized by centrifuging and resuspended in SDS-sample buffer. The supernatant proteins were separated by SDS-PAGE (polyacrylamide gel electrophoresis) in a 15% polyacrylamide gel and tested for expression and secretion after transfer to nitrocellulose with a polyclonal antibody, which is directed against the hemolysin portion of the fusion protein. B1 stands for the huZPB-1 construct (see Figure 8); B2 stands for the huZPB-2 construct (see Figure 8); B3 stands for the huZPB-3 construct (see Figure 8); B4 stands for the huZPB-4 construct (see Figure 8); B5 stands for the huZPB-5 construct (see Figure 8) and pM0 stands for the vector control.
Figure 7 shows a Western-blot, in which various pMOhlyl(hu)ZPC/constructs were tested for expression and secretion. After transformation of Yersinia enterocolitiaa Wap Yv-515 with the various pM0/(hu)ZPC/constructs, 2 ml of supernatant from overnight cultures was precipitated with 10%
(v/v) TCA (trichloroacetic acid) for 4 hours on ice, pelletized by centrifuging and resuspended in SDS-sample buffer. The supernatant proteins were separated by SDS-PAGE (polyacrylamide gel electrophoresis) in a 15% polyacrylamide gel and tested for expression and secretion after transfer to nitrocellulose with a polyclonal antibody, which is directed against the hemolysin portion of the fusion protein. C1 stands for the huZPC-1 construct (see Figure 12); C2d stands for the huZPC-2 construct with a double insert (see Figure 12); C3d stands for the huZPC-3 construct with a double insert (see Figure 12); C4 stands for the huZPC-4 construct (see Figure 12), and.pMO stands for the vector control.
Figure 8 shows a gene map of the cDNA of huZPA. The arrows show the positions of the primer that is selected for a PCR
amplification (Table 1), whereby the numbers indicate the starting point on the 5'-end of the primer relative to the starting codon of the cDNA sequence of the gene. The solid rectangles show the amino-terminal transport signal, the putative fusion gap site and a strongly hydrophobic area on the C-terminus that could act as a membrane anchor. The fragments that are amplified by PCR are depicted in the lower part with starting and ending points, which are produced by an Nsi-I restriction endonuclease digestion of the PCR products and henceforth referred to as huZPA-1, huZPA-2, etc. In addition, the length of the fragments is indicated both in base pairs (bp) and in amino acids (aa). The calculated molecular weights are reproduced for the zona fragments and in parentheses for the zona/HlyA fusion proteins in kDA. Finally, it is indicated whether a secretion of the fusion proteins with an antibody that is directed against the HlyA portion was detected.
Figure 9 shows a gene map of the CDNA of huZPB. The arrows show the positions of the primer that is selected for a PCR
amplification (Table 1), whereby the numbers indicate the starting point on the 5'-end of the primer relative to the starting codon of the cDNA sequence of the gene. The solid rectangles show the amino-terminal transport signal, the putative fusion gap site and a strongly hydrophobic area on the C-terminus that could act as a membrane anchor. The fragments that are amplified by PCR are depicted in the lower part with starting and ending points, which are produced by an Nsi-I restriction endonuclease digestion of the PCR products and henceforth referred to as huZPB-1, huZPB-2. In addition, the length of the fragments is indicated both in base pairs (bp) and in amino acids (aa). The calculated molecular weights are reproduced for the zona fragments and in parentheses for the zona/HlyA fusion proteins in kDa. Finally, it is indicated whether a secretion of the fusion proteins with an antibody that is directed against the HlyA portion was detected.
Figure 10 shows a gene map of cDNA of huZPC. The arrows show the positions of the primer that is selected for a PCR
amplification (Table 1), whereby the numbers indicate the starting point on the 5'-end of the primer relative to the starting codon of the cDNA sequence of the gene. The solid rectangles show the amino-terminal transport signal, the putative fusion gap site and a strongly hydrophobic area on the C-terminus that could act as a membrane anchor. The fragments that are amplified by PCR are depicted in the lower part with starting and ending points, which are produced by an Nsi-I restriction endonuclease digestion of the PCR products and henceforth referred to as huZPC-1, huZPC-2, etc. In addition, the length of the fragments is indicated both in base pairs (bp) and in amino acids (aa). The calculated molecular weights are reproduced for the zona fragments and in parentheses for the zona/HlyA fusion proteins in kDa. Finally, it is indicated whether a secretion of the fusion proteins with an antibody that is directed against the HlyA portion was detected.
Figure 11 shows a gene map of the cDNA of mZPB. The arrows show the positions of the primer that is selected for a PCR
amplification (Table 1), whereby the numbers indicate the starting point on the 5'-end of the primer relative to the starting codon of the cDNA sequence of the gene. The solid rectangles show the amino-terminal transport signal, the putative fusion gap site and a strongly hydrophobic area on the C-terminus that could act as a membrane anchor. The fragments that are amplified by PCR are depicted in the lower part with starting and ending points, which are produced by an Nsi-I restriction endonuclease digestion of the PCR products and henceforth referred to as mZPB-1, huZPB-2, etc. In addition, the length of the fragments is indicated both in base pairs (bp) and in amino acids (aa). The calculated molecular weights are reproduced for the zona fragments and in parentheses for the zona/HlyA fusion proteins in D. Finally, it is Figure 12 shows a gene map of cDNA of huZPC. The arrows show the positions of the primer that is selected for a PCR
amplification (Table 1), whereby the numbers indicate the starting point on the 5~-end of the primer relative to the starting codon of the cDNA sequence of the gene. The fragments that are amplified by PCR are depicted in the lower part with starting and ending points, which are produced by Nsi-I
restriction endonuclease digestion of the PCR products and henceforth referred to as huZPC-1, huZPC-2, etc. In addition, the length of the fragments is indicated both in base pairs (bp) and in amino acids (aa). The calculated molecular weights are reproduced for the zona fragments and in parentheses for the zona/HlyA fusion proteins in kDa. Finally, it is indicated whether a secretion of the fusion proteins with an antibody that is directed against the HlyA portion was detected.
The assumption is that one skilled in the art can execute this invention to its full scope based on the description. Many known techniques and protocols for manipulating nucleic acids, such as, for example, metagenesis, sequencing, the introduction of nucleic acid in cells or the analysis of proteins is in detail in Short Protocols in Molecular Biology, Second Edition, Ausrubel et al. Eds., John Wiley & Sons, 1992; Molecular Cloning: A
Laboratory Manual: 2nd Edition, Sambrock~et al., 1989, Cold Spring Harbor Laboratory Press. The content of Sambrook et al.
and Ausrubel et al. is incorporated herewith as reference.
This invention contains pharmaceutical preparations for all uses and the methods that are described. The invention thus contains, for example, pharmaceutical preparations that contain the attenuated bacteria with the secretion vector according to this invention. The oral administration is carried out preferably in the form of encapsulated, biodegradable polymers, for example PLPG.
The following examples are used only for a more detailed description and show that the invention is feasible, and the examples are in no way to act in a limiting manner.
Example 1 Cloning and Expression of huZPA, huZPB, huZPC and mZPB Fragments in E. aoli and 8. typhimurium The plasmids pGEX-KG-huZPA, pGEX-KG-huZPB and pGEX-KG-huZPC
(Peter Bringmann, Schering AG), which carry the cDNA of the human ZPA, ZPB and ZPC gene and the ovarian mRNA of mice, which made possible the cDNA synthesis of the mouse ZPB-gene, are the starting point of the cloning strategy. Starting from mRNA and isolated from the ovaries of superovulating mice (Uwe Eberspacher, Schering AG), the corresponding CDNA was synthesized as follows: about 5 ~g of RNA was mixed in 32 ~C1 of DEPC-H20 with 3 ~,l of oligo-dT-primer and incubated for 5 minutes at 65°C.
The batch is allowed to cool for 10 minutes at room temperature, and the following reagents are added: 5 ~,1 of synthesis buffer, ~1 of 0.1 M DTT, 1 ~C1 of RNase inhibitor, 3 ul of 25 mmol of dNTPs and 1 ~,1 of MMLV reverse transcriptase (20 U/~C1) (1st Strand Synthesis Kit, Stratagene). An incubation is carried out for 1 hour at 37°C. With the aid of specific oligonucleotides (Table 1), gene fragments that overlap by PCR from the corresponding cDNA were amplified, which have 5' and 3' Nsi I-restriction interfaces (Figures 8 to 12: maps of the ZP-genes with indication of the primer positions and the gene fragments that result from the above). The PCR-amplification (Saiki et ~al., 1988) was performed in a Thermocycler 60/2 (bio-med, Theres, Germany). For this purpose, cDNA was amplified with the corresponding 5'- and 3'-primers (1 ~mol each of final concentration), dNTPs (200 ~,mol each) and 2.5 U Taq DNA-polymerase (Promega) and the buffer of the manufacturer in a volume of 100 ~,1. Before the first cycle, a denaturation took place for 3 minutes at 91°C. 30 cycles then followed under the following conditions: 1 minute of denaturation at 91°C, 1 minute of annealing at 55°C and 1 minute of extension at 72°C. The reaction products were electrophoretically separated in a 2%
agarose gel and made visible by coloration with ethidium bromide.
These PCR products were first inserted "blunt" into the Sma I-interface of the vector pUCl8. The ZP-fragment was then cut out by Nsi I-digestion and inserted into the expression vector pMOhlyi (Figure 1). After transformation of E. coli DHSa with the various pMOhlyl/ZP-constructs, 2 ml of supernatant from overnight cultures was precipitated with 10% (v/v) TCA for 4 hours on ice, pelletized by centrifuging and resuspended in the SDS-sample buffer. The supernatant proteins were separated by SDS-PAGE in a 15% polyacrylamide gel and tested for the secretion of zona/hemolysin-fusion proteins after transfer to nitrocellulose. With the aid of a polyclonal antibody, which is directed against the hemolysin portion of the fusion protein, the expression and secretion of two huZPA fragments (A1 and A4, Figure 2) and four huZPB-fragments (B1-B4, Figure 3) could be detected. For huZPC, a transport-competent domain was identified (C1, Figure 4). Analogously to huZPB, the secretion of three protein fragments from mZPB could also be shown by the transport system (B1-B3, Figure 5).
Then, the transformation of the described pMOhly1 derivatives took place in the 8. typhimurium LB5000 strain, in which the restriction system is absent, so that E. coli-DNA can be introduced efficiently in this strain. After the recombinant expression vectors were isolated from 8. typhimurium LB5000, it was possible to transform the 8. typhimurium SL7207 strain successfully. As already described several times, this strain is suitable as an inoculation strain. The secretion of the ZP/Hly-fusion proteins also is carried out in this strain. By way of example, Figure 6 shows the expression and the secretion of the huZPB fragments in salmonella. After oral administration in mice, these salmonella that express ZP/Hly should induce a mucosal immunity, which is also directed specifically against the ZP-portion of the fusion protein.
Example 2 Cloning and Expression of huZPC, huZPB, huZPC and mZPB Fragments in Y. enterocolitica The plasmids pGEX-KG-huZPA, pGEX-KG-huZPB and pGEX-KG-huZPC
(Peter Bringmann, Schering AG), which carry the cDNA of the human ZPA, ZPB and ZPC gene and the ovarian mRNA of mice, which made possible the cDNA synthesis of the mouse ZPB-gene, are the starting point of the cloning strategy. Starting from mRNA and isolated from the ovaries of superovulating mice (Uwe Ebersp~cher, Schering AG), the corresponding cDNA was synthesized as follows: about 5 ~Cg of RNA was mixed in 32 ~C1 of DEPC-H20 with 3 ~C1 of oligo-dT-primer and incubated for 5 minutes at 65°C.
The batch is allowed to cool for 10 minutes at room temperature, and the following reagents are added: 5 ~,1 of synthesis buffer, 5 ul of 0.1 M DTT, 1 ~1 of RNase inhibitor, 3 ~1 of 25 mmol of dNTPs and 1 ~1 of MMLV reverse transcriptase (20 U/~Cl) (1st Strand Synthesis Kit, Stratagene). An incubation is carried out for 1 hour at 37°C. With the aid of specific oligonucleotides (Table 1), gene fragments that overlap by PCR from the corresponding cDNA were amplified, which have 5' and 3' Nsi I-restriction interfaces (Figures 8 to 12: maps of the ZP-genes with indication of the primer positions and the gene fragments that result from the above). The PCR-amplification (Saiki et al., 1988) was performed in a Thermocycler 60/2 (bio-mad, Theres, Germany). For this purpose, cDNA was amplified with the corresponding 5'- and 3'-primers (1 ~mol each of final concentration), dNTPs (200 ~mol each) and 2.5 U Taq DNA-polymerase (Promega) and the buffer of the manufacturer in a volume of 100 ~1. Before the first cycle, a denaturation took place for 3 minutes at 91°C. 30 cycles then followed under the following conditions: 1 minute of denaturation at 91°C, 1 minute of annealing at 55°C and 1 minute of extension at 72°C. The reaction products were electrophoretically separated in a 2%
agarose gel and made visible by coloration with ethidium bromide.
These PCR products were first inserted "blunt" in the Sm I-interface of the vector pUCl8. The ZP-fragment was then cut out by Nsi I-digestion and -inserted in the expression vector pMOhlyi (Figure 1). After transformation of E. coli DHSa with the various pMOhlyl/ZP-constructs, 2 ml of supernatant from overnight cultures was precipitated with 10% (v/v) TCA for 4 hours on ice, palletized by centrifuging and resuspended in the SDS-sample buffer. The supernatant proteins were separated by SDS-PAGE in a 15% polyacrylamide gel and tested for the secretion of zona/hemolysin-fusion proteins after transfer to nitrocellulose.
With the aid of a polyclonal antibody, which is directed against the hemolysin portion of the fusion protein, the expression and secretion of huZPA fragments, huZPB-fragments and huZPC could be detected (see above). Then, the transformation of the described pMOhlyl derivatives took place in the Y. enteroaolitica LB5000 strain, in which the restriction system is absent, so that E.
cola-DNA can be introduced efficiently in this strain. After the recombinant expression vectors were isolated from Y.
enterocolitica LB5000, it was possible to transform the Y.
enterocolitica Wap Yv-515 strain successfully. As already described several times, this strain is suitable as an inoculation strain. The secretion of the ZP/Hly-fusion proteins also is carried out in this strain. By way of example, Figure 7 shows the expression and the secretion of the huZPC fragments in Y. enterocolitica. After oral administration in mice, these yersinia that express ZP/Hly should induce a mucosal immunity, which is also directed specifically against the ZP-portion of the fusion protein.
Table 1: Primer sequences for the amplification of ZP-gene fragments by PCR and expected PCR-product values [Key:] PCR-Produkt = PCR product ZP-Fragment PCR-Produkt (bp) huZPA
huZPA1 5'-GAAATGGCATGCATGCATCTGTG-3'390 5'-GCTGAAGCATGCATTCATGGCCTC-3' huZPA2 5'-CTGCCAGATGCATTGAAGGAAGGC-3'408 ~
5'-GTCAGCTTGAATGCATCTAAGTAGAAC-3' ~ huZPA3 5'-CGAAATTATATGCATAATGCCTAC-3'456 !
I
_ _ _ 5'-GAGTAAGGCATGCATCGTTGATG-3' huZPA4 5'-GTGAAGTATGCATATAGCAGG-3' 318 _ _ 5'-CATATGCACATGCATCCACGACAAC-3' huZPA5 5'-CACCATGGATGCATACTCTTTCC-3'249 5'-GCCTAGAGGATGCATGGCAGGTCAG3' huZPB
huzPB1 5'-GCTTCCAGTATGCATTAAACCTC-3'207 5'-CGCGCCTGATGCATCAACTCCAAC-3' huZPB2 5-GTGGTGTfGGATGCATCCTATAGC-3'399 5'-CAGGGTTACATGCATfGTCATTCC-3' huZPB3 5'-CTCTfGGATGCATTGCGCTTGGCC-3'375 5'-CTTCACCAATGCATAGTCACCAAC-3' huZPB4 5'-GAGACCCAGCATGCATCCCTCACTC-f498 5-GCCTTTGCTAGATGCATTAGCAGTAG-3' huZPBS 5'-GCCAGCCTGCTGATGCATCATCCTG-3'213 5'-CAGCCAAGTAGGATGCATACAAGGC-3' ZP-Fragment PCR-Produkt ~ (bp) huZPC
I huZPC1 5'-GCCAGCCATGCATAGACGTCCGTAC-3'495 ~ ~
- -5'-CAGTGTGGATTT_ATGC_ATGGAGGTG-3' i huZPC2 5'-GAGAACTGGAATGCATAGAAGAGG-3'366 ~
5'CCAGCTGTTGGATGCATTGCTGAAG-3' huZPC2' ~ 5-CAATGTGAGCAGCCATGCATTCCTGC-3'~ 285 ' - - i 5'-GAATGCAGAAGA_TGCATCAGTGAG-3' i huZPC3 ~ 5'-CAGGACCCAGATGCATTCAACAAG-3'~ 363 - -5'-GAGAC~C~G 1 C~GGA I GGP. r I Gi.:GAGAC:-3' ' i I
I h Z C3' S'-GACCAGAATGCATCCCCTTATCAG3'360 ~
5'-GGTTACGGGATGCATACCTGGAC-3', I
~
huZPC4 5'GCCAGCCATGCATATACGTCCGTAC-3'936 ~
5'-GGTTACGGGATGCATACCTGGAG3' mZPB
mZPB1 ~ 5'-GAATACAGCTATGCATGTGGGGTAC-3'450 ~
5'-GCAGGAGATGCATGGATGAAGCTC-3' mZP82 5'-CACACCTTGGATGCATCTGGCCAG3'408 5'-CATCCGTTGGATGCATAGGCCAG-3' mZPB3 5'-GCCTfGACACATG_CA_TTCCTGCTAG-3'339 5'-CTTATCCGATGCATTCCTCAGCTG3' mZPB4 5'-GTGACTCAATATGCATCCCTGAGGC-3'393 5'-CAGAGGGTGGCATGCATAGGCACTAC-3' Underlined bases indicate a deviation from the respective ZP-sequence to introduce the Nsi I interface that is necessary for cloning
Claims (6)
1. Use of a secretion vector in which (a) genes or gene fragments that are suitable for birth control are inserted, which (b) contains the entire hemolysin operon including the hly-specific promoter and an enhancer-similar regulator hlyR and in which (c) a major part of the hlyA gene was deleted, whereby (d) the proteins or protein fragments that are suitable for birth control are synthesized, and (e) by the secretion of these antigens by attenuated salmonella or other gram-negative attenuated inoculation strains, (f) an oral vaccination is produced.
2. Use according to claim 1, whereby genes or gene fragments that are suitable for birth control code for zona pellucida proteins.
3. Use according to claim 1, whereby the secretion vector is pMOhly1.
4. Use according to claim 1, whereby the salmonella strain is Salmonella typhimurium.
5. Use according to claim 1, whereby the strain is Yersinia enterocolitica.
6. Process for birth control by using a secretion vector in which (a) genes or gene fragments that are suitable for birth control are inserted, which (b) contains the entire hemolysin operon including the hly-specific promoter and an enhancer-similar regulator hlyR, and in which (c) a major part of the hlyA gene was deleted, whereby (d) the proteins or protein fragments that are suitable for birth control are synthesized, and (e) by the secretion of these antigens by attenuated salmonella or other gram-negative attenuated inoculation strains, (f) an oral vaccination is produced.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19720761A DE19720761A1 (en) | 1997-05-07 | 1997-05-07 | Process for the synthesis and secretion of proteins or protein fragments suitable for contraception by attenuated Salmonella or another Gram-negative attenuated vaccine strain to produce an oral vaccination |
| DE19720761.8 | 1997-05-07 | ||
| PCT/EP1998/002679 WO1998050067A2 (en) | 1997-05-07 | 1998-05-07 | Use or a secretion vector for fertility control by oral vaccination |
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| Publication Number | Publication Date |
|---|---|
| CA2288602A1 true CA2288602A1 (en) | 1998-11-12 |
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ID=7829783
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002288602A Abandoned CA2288602A1 (en) | 1997-05-07 | 1998-05-07 | Use of a secretion vector for fertility control by oral vaccination |
Country Status (18)
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| EP (1) | EP1015023A1 (en) |
| JP (1) | JP2002501507A (en) |
| KR (1) | KR20010012257A (en) |
| CN (1) | CN1255067A (en) |
| AU (1) | AU7910798A (en) |
| BG (1) | BG103850A (en) |
| BR (1) | BR9809244A (en) |
| CA (1) | CA2288602A1 (en) |
| DE (1) | DE19720761A1 (en) |
| EE (1) | EE9900516A (en) |
| HU (1) | HUP0002752A2 (en) |
| IL (1) | IL132521A0 (en) |
| IS (1) | IS5199A (en) |
| NO (1) | NO995429L (en) |
| PL (1) | PL336786A1 (en) |
| SK (1) | SK151799A3 (en) |
| TR (1) | TR199902671T2 (en) |
| WO (1) | WO1998050067A2 (en) |
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| DE10206325A1 (en) * | 2002-02-14 | 2003-09-04 | Medinnova Ges Med Innovationen | Coated microorganism |
| US6989160B2 (en) * | 2002-03-19 | 2006-01-24 | Council Of Scientific And Industrial Research | Therapeutic/edible compositions comprising herbal ingredients and methods for treating hyperglycemia |
| CN101175859B (en) * | 2005-05-12 | 2013-04-03 | 味之素株式会社 | Method for producing protein |
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| CA2096529C (en) * | 1990-11-21 | 2003-01-21 | Roy Curtiss, Iii | Recombinant avirulent salmonella antifertility vaccines |
| US5480799A (en) * | 1993-12-10 | 1996-01-02 | The University Of North Carolina At Chapel Hill | Sperm antigen corresponding to a sperm zona binding protein autoantigenic epitope |
-
1997
- 1997-05-07 DE DE19720761A patent/DE19720761A1/en not_active Withdrawn
-
1998
- 1998-05-07 EP EP98929286A patent/EP1015023A1/en not_active Withdrawn
- 1998-05-07 SK SK1517-99A patent/SK151799A3/en unknown
- 1998-05-07 CA CA002288602A patent/CA2288602A1/en not_active Abandoned
- 1998-05-07 KR KR1019997010208A patent/KR20010012257A/en not_active Application Discontinuation
- 1998-05-07 AU AU79107/98A patent/AU7910798A/en not_active Abandoned
- 1998-05-07 BR BR9809244-8A patent/BR9809244A/en not_active IP Right Cessation
- 1998-05-07 JP JP54774098A patent/JP2002501507A/en active Pending
- 1998-05-07 HU HU0002752A patent/HUP0002752A2/en unknown
- 1998-05-07 EE EEP199900516A patent/EE9900516A/en unknown
- 1998-05-07 WO PCT/EP1998/002679 patent/WO1998050067A2/en not_active Application Discontinuation
- 1998-05-07 IL IL13252198A patent/IL132521A0/en unknown
- 1998-05-07 PL PL98336786A patent/PL336786A1/en unknown
- 1998-05-07 TR TR1999/02671T patent/TR199902671T2/en unknown
- 1998-05-07 CN CN98804817A patent/CN1255067A/en active Pending
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| Publication number | Publication date |
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| EE9900516A (en) | 2000-06-15 |
| AU7910798A (en) | 1998-11-27 |
| JP2002501507A (en) | 2002-01-15 |
| WO1998050067A9 (en) | 1999-03-25 |
| IL132521A0 (en) | 2001-03-19 |
| BG103850A (en) | 2000-05-31 |
| WO1998050067A2 (en) | 1998-11-12 |
| BR9809244A (en) | 2000-06-27 |
| KR20010012257A (en) | 2001-02-15 |
| HUP0002752A2 (en) | 2000-12-28 |
| IS5199A (en) | 1999-09-28 |
| SK151799A3 (en) | 2000-06-12 |
| TR199902671T2 (en) | 2000-09-21 |
| NO995429D0 (en) | 1999-11-05 |
| PL336786A1 (en) | 2000-07-17 |
| NO995429L (en) | 2000-01-07 |
| CN1255067A (en) | 2000-05-31 |
| DE19720761A1 (en) | 1998-11-12 |
| EP1015023A1 (en) | 2000-07-05 |
| WO1998050067A3 (en) | 2000-06-29 |
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