Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/0005—Vertebrate antigens
  • A61K39/0006—Contraceptive vaccins; Vaccines against sex hormones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
  • A61P15/16—Masculine contraceptives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
  • A61P15/18—Feminine contraceptives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
  • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
  • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

• the invention relates to a method for fertility control by oral vaccination using attenuated Salmonella or other Gram-negative attenuated vaccine strains using various expression systems which allow the generation of a targeted MHCII / CD4 or MHCI / CDg immune response.
• the antigen characterized by Moore and Lee as well as the SP-10 immunogen were described by the "World Health Organization" (special area for vaccines for fertility regulation) as primary vaccine candidates (Anderson et al., 1987).
• the three protein components of the zona pellucida (ZP), an extracellular matrix that surrounds mammals, are also suitable as antigens. These proteins are usually referred to as ZPA, ZPB and ZPC (Wassarmann, 1987, Science 235, 553-560).
• ZPA zona pellucida
• ZPB ZPC
• the egg-sperm interaction is to be blocked or influenced by an immunological blockade.
• the zona protein which is responsible for the initial binding of the sperm to the zona, have also confirmed this.
• (a) contains the complete hemolysin operon including the hly-specific promoter and an enhancer-like regulator hlyR and in which
• the genes or gene fragments suitable for fertility control encode zona pellucida proteins. In a preferred embodiment of the invention, the genes or gene fragments suitable for fertility control encode zona pellucida proteins.
• the secretion vector is pMOhlyl.
• the Salmonella strain is Salmonella typhimurium.
• the genes or gene fragments suitable for fertility control code for zona pellucida proteins, the secretion vector is pMOhlyl and the Salmonella strain is Salmonella typhimurium.
• Genes or gene fragments suitable for fertility control are understood to mean all genes or gene fragments which code for proteins or protein fragments suitable for fretility control.
• HlyA Escherichia coli hemolysin
• HlyAg transport signal
• the E. coli hemolysin secretion system is made up of three membrane proteins. Two of these proteins, HlyB (Gentschev & Goebel, 1990) and HlyD (Schülein et al, 1992), are located in the inner membrane and are encoded by genes that are part of the hemolysin determinant. This consists of an operon including the four genes hlyC, hlyA, hlyB and hlyD (Wagner et al, 1983; Hess et al., 1986). The third protein of the translocation system, TolC, is located in the outer membrane (Wandersman & Delepelaire, 1990).
• HlyAg is fused with other proteins or protein fragments to secrete these fusion proteins with the aid of the hemolysin secretion system (Blight & Holland 1994; Gentschev et al., 1994).
• the secretion efficiency depends on the folding and conformation of the reporter protein.
• the E. coli hemolysin secretion system is functional in attenuated Salmonella, which act as vaccine strains, and can be used to export fusion proteins (Gentschev et al, 1992; Su et al., 1992).
• the genetic systems used for this have so far been based on two components: a plasmid which carries the genes necessary for transport [hlyB and hlyD) 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-like regulator hlyR (Vogel et al, 1988).
• HlyAg carboxy terminal amino acids
• the genetic information for antigens with a size of 10-1000 amino acids can be inserted into this secretion vector pMOhlyl, which enables the secretion of these antigens in attenuated Salmonella and other Gram-negative attenuated vaccine strains (e.g. BEcoli, Vibrio cholerae, Yeresina enterocoliticä).
• attenuated Salmonella and other Gram-negative attenuated vaccine strains e.g. BEcoli, Vibrio cholerae, Yeresina enterocoliticä.
• the transport of heterologous fusion proteins is made possible by a single plasmid.
• hemolysin secretion apparatus in comparison to transport systems previously used for an antigen presentation, which are only suitable for the transport of usually only relatively short peptides to the outside of the bacterial cell (Cardenas & Clements, 1992) is the significantly more variable size of the transport-competent proteins.
• the same antigen can be presented in suitable attenuated Gram-negative vaccine strains in a cytoplasmic, surface-bound or in secreted form.
• listeriolysin from Listeria monocytogenes
• Salmonella vaccine strain By using listeriolysin (from Listeria monocytogenes) made secretable in Salmonella, it can additionally be achieved that the same antigen is processed in the phagosome or, to a greater extent, in the cytosol of the antigen-presenting macrophage cell after infection with the antigen-producing Salmonella vaccine 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).
• cytokines and soluble cytokine receptors are also secreted separately or in combination with the antigen to be produced in attenuated Salmonella (Schülein, 1993; Gentschev, unpublished), the immune response (e.g. towards TJJI or Tpj2) can also be varied.
• the efficient secretion of proteins or protein fragments / HlyAs fusions suitable for fertility control and the high stability of the vector enable a completely new, highly specific contraceptive vaccination with the help of attenuated Salmonella or other Gram-negative attenuated vaccine strains.
• the antigen proteins or protein fragments suitable for fertility control
• the antigen becomes accessible to the host's immune system by secretion of the proteins or protein fragment / HlyAg fusions suitable for fertility control and, depending on the protein epitope and the secretion pathway, leads to activation of B and / or T cells.
• HlyAs is a weak antigen for B and T cells
• HlyAg fusions induce antibodies and T cells that are mainly directed against the part of the reporter antigen.
• Attenuated Salmonella and Yersinia strains that secrete antigens are particularly suitable for inducing a humoral, mucosal immune response and are therefore preferred.
• Induction of mucosal immunity also results in the production of antigen-specific antibodies to distant mucosal Surfaces. Therefore, a B cell response at the site of infection also stimulates mucosal immunity in the reproductive tract.
• Figure 1 shows a vector map of the secretion plasmid pMOhlyl.
• Figure 2 shows a Western blot in which various pMOhlyl / (hu) ZPA / constructs were tested for expression and secretion.
• the supernatant proteins were separated by SDS-PAGE (polyacrylamide gel electrophoresis) in a 15% polyacrylamide gel and, after transfer to nitrocellulose with a polyclonal antibody directed against the hemolysin portion of the fusion protein, tested for expression and secretion.
• AI stands for the huZPA-1 construct (see Figure 7)
• A4 stands for the huZPA-4 construct (see Figure 7)
• pMO stands for the vector control
• 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.
• Bl 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)
• pMO stands for vector control.
• Figure 4 shows a Western blot in which various pMOhlyl ⁇ hu) ZPC / constructs were tested for expression and secretion.
• the supernatant proteins were separated by SDS-PAGE (polyacrylamide gel electrophoresis) in a 15% polyacrylamide gel and, after transfer to nitrocellulose with a polyclonal antibody directed against the hemolysin portion of the fusion protein, tested for expression and secretion.
• Cl 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)
• pMO stands for vector control .
• Figure 5 shows a Western blot in which various pMOhlyl / (m) ZPB / constructs were tested for expression and secretion.
• the supernatant proteins were separated by SDS-PAGE (polyacrylamide gel electrophoresis) in a 15% polyacrylamide gel and after transfer to nitrocellulose with a polyclonal Antibody directed against the hemolysin portion of the fusion protein tested for expression and secretion.
• Bl 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)
• pMO stands for vector control.
• Figure 6 shows a Western blot in which various pMOhlyl (hu) ZPB constructs were tested for expression and secretion.
• S. typhimurium SL7207 After transformation of S. typhimurium SL7207 with the various pMO / (hu) ZPB constructs, 2 ml of supernatant culture supernatant with 10% (v / v) TCA (trichloroacetic acid) was precipitated on ice for 4 h, pelleted by centrifugation and in SDS sample buffer resuspended.
• TCA trichloroacetic acid
• the supernatant proteins were separated by SDS-PAGE (polyacrylamide gel electrophoresis) in a 15% polyacrylamide gel and, after transfer to nitrocellulose, tested for expression and secretion with a polyclonal antibody which is directed against the hemolysin portion of the fusion protein.
• Bl 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)
• pMO stands for vector control.
• Figure 7 shows a Western blot in which various pMOhlyl (hu) ZPC / constructs were tested for expression and secretion.
• 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 directed against the hemolysin portion of the fusion protein.
• Cl 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 double insert (see Figure 12)
• C4 stands for the huZPC-4 construct (see Figure 12)
• pMO stands for the vector control.
• Figure 8 shows a gene map of the cDNA from huZPA.
• the arrows show the positions of the primers selected for PCR amplification (Table 1), the numbers indicating the starting point at the 5 ' end of the primer in relation to the start codon of the cDNA sequence of the gene.
• the filled rectangles show the amino terminal transport signal, the putative fusion cleavage site and a strongly hydrophobic area at the C-terminus that could act as a membrane anchor.
• the fragments amplified by PCR are shown in the lower part with the start and end point, which result from an Nsi-I restriction endonuclease digestion of the PCR products, and are designated continuously with huZPA-1, huZPA-2 etc.
• the length of the fragments is given both in base pairs (bp) and in amino acids (aa).
• the calculated molecular weights are given in kDa for the zona fragments and in brackets for the zona / HlyA fusion proteins. Finally, it is indicated whether secretion of the fusion proteins was detected with an antibody directed against the HlyA portion.
• Figure 9 shows a gene map of the CDNA from huZPB.
• the arrows show the positions of the primers selected for PCR amplification (Table 1), the numbers indicating the starting point at the 5 'end of the primer in relation to the start codon of the cDNA sequence of the gene.
• the filled rectangles show the amino terminal transport signal, the putative fusion cleavage site and a strongly hydrophobic area at the C-terminus that could act as a membrane anchor.
• the fragments amplified by PCR are shown in the lower part with the start and end point, which result from an Nsi-I restriction endonuclease digestion of the PCR products, and are continuously designated with huZPB-1, huZPB-2 etc.
• the length of the fragments is given both in base pairs (bp) and "in amino acids (aa).
• the calculated molecular weights are given in kDa for the zona fragments and in brackets for the zona / HlyA fusion proteins indicated whether a secretion of the fusion proteins with an antibody directed against the HlyA portion was detected.
• Figure 10 shows a gene map of the cDNA from huZPC.
• the arrows show the positions of the primers selected for PCR amplification (Table 1), the numbers indicating the starting point at the 5 ' end of the primer in relation to the start codon of the cDNA sequence of the gene.
• the filled rectangles show the amino terminal transport signal, the putative fusion cleavage site and a strongly hydrophobic area at the C-terminus, which could act as a membrane anchor.
• the fragments amplified by PCR are shown in the lower part with the start and end point, which result from an Nsi-I restriction endinuclease digestion of the PCR products, and are continuously designated with huZPC-1, huZPC-2 etc.
• the length of the fragments is given both in base pairs (bp) and in amino acids (aa).
• the calculated molecular weights are given in kDa for the zona fragments and in brackets for the zona / HlyA fusion proteins. Finally, it is indicated whether secretion of the fusion proteins was detected with an antibody directed against the HlyA portion.
• Figure 11 shows a gene map of the cDNA from mZPB.
• the arrows show the positions of the primers selected for PCR amplification (Table 1), the numbers indicating the starting point at the 5 'end of the primer in relation to the start codon of the cDNA sequence of the gene.
• the filled rectangles show the amino terminal transport signal, the putative fusion cleavage site and a strongly hydrophobic area at the C-terminus, which could act as a membrane anchor.
• the fragments amplified by PCR are shown in the lower part with the start and end point, which result from an Nsi-I restriction endonuclease digestion of the PCR products, and are designated continuously with mZPB-1, huZPB-2 etc.
• Figure 12 shows a gene map of the cDNA from huZPC.
• the arrows show the positions of the primers selected for PCR amplification (Table 1), the numbers indicating the starting point at the 5 ' end of the primer in relation to the start codon of the cDNA sequence of the gene.
• the fragments amplified by PCR are shown in the lower part with the start and end point, which result from an Nsi-I restriction endinuclease digestion of the PCR products, and are designated continuously with huZPC-1, huZPC-2 etc. Furthermore, the length of the fragments is given both in base pairs (bp) and in amino acids (aa). The calculated molecular weights are given in kDa for the zona fragments and in brackets for the zona / HlyA fusion proteins. Finally, it is indicated whether secretion of the fusion proteins was detected with an antibody directed against the HlyA portion.
• the present invention includes pharmaceutical preparations for all uses and methods described.
• the invention includes pharmaceutical preparations which contain the attenuated bacteria with the secretion vector according to the present invention.
• Oral administration is preferably carried out in the form of encapsulated biodegradable polymers, for example PLPG.
• the starting point of the cloning strategy are 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 genes and ovarian mRNA of the mouse which enabled the cDNA synthesis of the mouse ZPB gene.
• pGEX-KG-huZPA pGEX-KG-huZPB
• pGEX-KG-huZPC Peter Bringmann, Schering AG
• RNA 5 ⁇ g RNA were mixed in 32 ⁇ l DEPC-H2O with 3 ⁇ l oligo-dT primer and for 5 min at 65 ° C incubated. The mixture is allowed to cool for 10 min at room temperature and the following reagents are added: 5 ⁇ l synthesis buffer, 5 ⁇ l 0.1 M DTT, 1 ⁇ l RNase inhibitor, 3 ⁇ l 25 mM dNTPs and 1 ⁇ l MMLV reverse transcriptase (20 U / ⁇ l) ( Is Strand Synthesis Kit, Stratagene). This is followed by an incubation for 1 h at 37 ° C.
• cDNA was amplified with the corresponding 5 'and 3' primers (each 1 ⁇ M final concentration), dNTPs (each 200 ⁇ M) and 2.5 U Taq DNA polymerase (Promega) and the manufacturer's buffer in a volume of 100 ⁇ l.
• denaturation was carried out for 3 min at 91 ° C. This was followed by 30 cycles under the following conditions: 1 min denaturation at 91 ° C, 1 min annealing at 55 ° C and 1 min extension at 72 ° C.
• the reaction products were separated electrophoretically in a 2% agarose gel and visualized by staining with ethidium bromide.
• the pMOhlyl derivatives described were then transformed into the S. typhimurium LB5000 strain, which lacks the restriction system, so that E. coli DNA can be introduced efficiently into this strain.
• the strain S. typhimurium SL7207 was successfully transformed. As already described several times, this strain is suitable as a vaccination strain.
• the ZP / Hly fusion proteins are also secreted in this strain.
• Figure 6 shows an example of the expression and secretion of the huZPB fragments in Salmonella. These salmonella expressing ZP / Hly should induce mucosal immunity in mice after oral administration, which is also specifically directed against the ZP portion of the fusion protein.
• Example 2 shows an example of the expression and secretion of the huZPB fragments in Salmonella. These salmonella expressing ZP / Hly should induce mucosal immunity in mice after oral administration, which is also specifically directed against the ZP portion of the fusion protein.
• the starting point of the cloning strategy are 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 genes and ovarian mRNA of the mouse which enabled the cDNA synthesis of the mouse ZPB gene.
• pGEX-KG-huZPA pGEX-KG-huZPB
• pGEX-KG-huZPC Peter Bringmann, Schering AG
• 5 ⁇ g RA were mixed in 32 ⁇ l DEPC-H2O with 3 ⁇ l oligo-dT primer and for 5 min at 65 ° C incubated. The mixture is allowed to cool for 10 min at room temperature and the following reagents are added: 5 ⁇ . Synthesis buffer, 5 ⁇ l 0 1 M DTT, 1 ⁇ l RNase inhibitor, 3 ⁇ l 25 ⁇ iM dNTPs and 1 ⁇ l MMLV reverse transcriptase (20 U / ⁇ l) (is Strand Synthesis Kit, Stratagene). This is followed by an incubation for 1 h at 37 ° C.
• cDNA was amplified with the corresponding 5 'and 3' primers (each 1 ⁇ M final concentration), dNTPs (each 200 ⁇ M) and 2.5 U Taq DNA polymerase (Promega) and the manufacturer's buffer in a volume of 100 ⁇ l.
• denaturation was carried out for 3 min at 91 ° C. This was followed by 30 cycles under the following conditions: 1 min denaturation at 91 ° C, 1 min annealing at 55 ° C and 1 min extension at 72 ° C.
• the reaction products were separated electrophoretically in a 2% agarose gel and visualized by staining with ethidium bromide.
• huZPA fragments huZPB fragments and hu ZPC could be detected with the help of a polyclonal antibody, which is directed against the hemolysin portion of the fusion protein (see above).
• the pMOhlyl derivatives described were then transformed into the Y. enterocolitica LB5000 strain, which lacks the restriction system, so that E. coli ONA can be efficiently introduced into this strain.
• the strain Y. enterocolitica WapYv-515 was successfully transformed. As already described several times, this strain is suitable as a vaccination strain.
• the ZP / Hly fusion proteins are also secreted in this strain.
• Figure 7 shows an example of the expression and secretion of the huZPC fragments in Y. enterocolitica. These ZP / Hly-expressing Yersinia should induce mucosal immunity in mice after oral administration, which is also specifically directed against the ZP portion of the fusion protein.
• Underlined bases indicate a deviation from the respective ZP sequence in order to introduce the Nsi I interface necessary for cloning.

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