DE19720761A1 - 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 - Google Patents

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

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 / CD ₄ or MHCI / CD ₈ immune response.

State of the art

It has long been known that women and men have significantly high antibody titers are often infertile to human sperm or have reduced fertility (Ingerslev and Ingerslev, 1989; Chen and Jones, 1981; Menge et al., 1982; Bronson et al., 1984). It was also shown to immunize female and male animals with Extracts of whole sperm can lead to induction of infertility (grief field and Foote, 1979; Munoz and Metz; 1978; Tung et al., 1979; Menge et al., 1979). Primakoff et al. (1988a) used a monoclonal antibody to target the guinea pig Isolate sperm-surface antigen PH-20 and could show that the injection of this purified antigen in female or male guinea pigs long-lasting immunization against fertility generated (1988b).

A number of other monoclonal antibodies directed against ejaculated human sperm or isolated sperm from other species have been shown to cross-react with human sperm. Some react with components of the seminal plasma and others recognize antigens of testicular origin. Monoclonal antibodies which immobilize or agglutinate human sperm or inhibit sperm binding and penetration of zonafree hamster oocytes have been described. Several human sperm antigens are currently known, such as the M _r 95,000 antigen (Moore et al., 1987); the 55 kDa antigen recognized by Lee's S36-37 mAbs (HSA-63) (Liu et al., 1990); and the human homolog of the sperm receptors of ZP-C (determined in the mouse by Saling and Bliel and Wassarman (Bliel, 1990; Leyton and Saling, 1989). Of further interest is the FA-1 antigen of the mouse and of humans, which is used for Was characterized (Naz, 1988) and the 24kDa antigen from rat testis and human testis (Shaha et al., 1990). The antigen characterized by Moore and Lee as well as the SP-10 immunogen were obtained from 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 called Designated ZPA, ZPB and ZPC (Wassarmann, 1987, Science 235, 553-560). The egg-sperm interaction should be blocked or influenced by an immunological block. Experiments with recombinantly produced This was also confirmed by the ZPC, the zona protein, which is responsible for the initial binding of the sperm to the zona. In all cases reported so far, however, irreversible damage to the ovary occurred with long-term immunization (Skinner et al., 1984, Endocrinology 115, 2418-2432). The mechanism of this loss of ovarian function has not yet been elucidated.

It has now been found that by inserting genes or gene fragments suitable for fertility control into a secretion vector, the

• (a) the complete hemolysin operon including the hly specific promoter and one Enhancer-like regulator hlyR contains and at which
• (b) a large part of the hlyA gene has been deleted,
• (c) synthesizing the proteins or protein fragments suitable for fertility control and
• (d) by the secretion of these antigens by attenuated Salmonella or other grief negative attenuated vaccine strains,
• (e) oral vaccination is generated.

In a preferred embodiment of the invention, those suitable for fertility control are encoded Genes or gene fragments for zona pellucida proteins.

In a further preferred embodiment, the secretion vector is pMOhly1.

In a further preferred embodiment, the Salmonella strain is Salmonella typhimurium.

In a particularly preferred embodiment, those suitable for fertility control are encoded Genes or gene fragments for zona pellucida proteins, the secretion vector is pMOhly1 and the Samonella strain is Salmonella typhimurium.

Genes or gene fragments suitable for fertility control include all genes or Genetic fragments understood that for proteins or suitable for fretility control Encode protein fragments.

Most proteins that are transported into the periplasm via the inner membrane of Gram-negative bacteria have an amino-terminal signal peptide that is split off during this sec-dependent transport process (Pugsley, 1993). In contrast, Escherichia coli hemolysin (HlyA) is secreted into the extracellular medium via the inner and outer membrane using a secretion system. In contrast to the classic N-terminal transport signal of sec-dependent protein transport, HlyA carries a transport signal (HlyA _S ) at the C-terminus, which is formed by 50-60 amino acids (Hess et al., 1990; Jarchau et al., 1994). This HlyA signal is not split off in the course of the secretion and hardly has any antigenic potential.

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). The translocation of HlyA across both membranes of Gram-negative bacteria requires energy in the form of ATP and a membrane potential of the inner membrane (Koronakis et al., 1995). In various cases it has already been shown that HlyA _{S is} fused with other proteins or protein fragments to secrete these fusion proteins using 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 pMOhly1 (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). A large part of the hlyA gene was deleted, so that only 34 amino-terminal and 61 carboxy-terminal amino acids (HlyA _S ) are encoded by HlyA. A unique Nsi I interface between the amino-terminal and carboxy-terminal residue of HlyA allows heterologous genes or gene fragments to be inserted in frame to HlyA _S. The genetic information for antigens with a size of 10-1000 amino acids can be inserted into this secretion vector pMOhly1, which enables the secretion of these antigens in attenuated Salmonella and other Gram-negative attenuated vaccine strains (e.g. BEcoli, Vibrio cholerae). In contrast to other secretion systems, the transport of heterologous fusion proteins is made possible by a single plasmid. Another important advantage of the hemolysin secretion apparatus compared to transport systems previously used for an antigen presentation, which are only suitable for the transport of mostly relatively short peptides to the outside of the bacterial cell (Cardenas & Clements, 1992), is the significantly more variable size of the transport-competent proteins.

By manipulating the HlyA secretion system, the same antigen can be presented in suitable attenuated Gram-negative vaccine strains in a cytoplasmic, surface-bound or secreted form. By using listeriolysin (from Listeria monocytogenes) made capable of secretion 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). Since this vector system could also be used to separate cytokines and soluble cytokine receptors separately or in combination with the antigen to be produced in attenuated Salmonella (Schülein, 1993; Gentschev, unpublished), the immune response (e.g. towards T _H1 or T _H2 ) vary additionally.

The efficient secretion of proteins or protein fragments / HlyA _S 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.

After oral administration of the recombinant Salmonella, the antigen (proteins or protein fragments suitable for fertility control) is accessible to the host's immune system by secretion of the proteins or protein fragment / HlyA _S fusions suitable for fertility control and, depending on the protein epitope and the secretion pathway, leads to the activation of B - and / or T cells. Since HlyA _{S is} a weak antigen for B and T cells, HlyA _S fusions induce antibodies and T cells that are mainly directed against the part of the reporter antigen. Attenuated Salmonella 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.

With the invention disclosed here, it is possible for the first time to use recombinant proteins or Parts of it without complex manufacturing processes for the production of a corresponding one To generate an immune response that leads to contraception directly in situ. With help corresponding proteins, these proteins or protein segments in phagosomes, be expressed intracellularly or extracellularly. With that, consciously humoral or cytotoxic immune responses are generated.

Description of the pictures

Fig. 1 shows a vector map of the secretion plasmid pMOhly1.

Fig. 2 shows a Western blot in which various pMOhly1 / (hu) ZPA / constructs were tested for expression and secretion. After transformation of E. coli DH5α with the various pMOhly1 / (hu) ZPA / constructs, 2 ml of supernatant from overnight cultures with 10% (v / v) TCA (trichloroacetic acid) were precipitated on ice for 4 h, pelleted by centrifugation and in SDS- Sample buffer resuspended. 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 which is directed against the hemolysin portion of the fusion protein, tested for expression and secretion. A1 stands for the huZPA-1 construct (see Fig. 7), A4 stands for the huZPA-4 construct (see Fig. 7), PMO stands for the vector control and B4 stands for the huZPB-4 construct (see Figure . 8).

Fig. 3 shows a Western blot in which various pMOhly1 / (hu) ZPB / constructs were tested for expression and secretion. After transformation of E. coli DH5α with the various pMOhly1 / (hu) ZPB / constructs, 2 ml of supernatant from overnight cultures with 10% (v / v) TCA (trichloroacetic acid) were precipitated on ice for 4 h, pelleted by centrifugation and in SDS- Sample buffer resuspended. 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 which is directed against the hemolysin portion of the fusion protein, tested for expression and secretion. B1 stands for the huZPB-1 construct (see Fig. 8), B2 stands for the huZPB-2 construct (see Fig. 8), B3 stands for the huZPB-3 construct (see Fig. 8), B4 stands for for the huZPB-4 construct (see Fig. 8), B5 stands for the huZPB-5 construct (see Fig. 8) and pMO stands for vector control.

Fig. 4 shows a Western blot in which various pMOhly1 / (hu) ZPC / constructs were tested for expression and secretion. After transformation of E. coli DH5α with the various pMOhly1 / (hu) ZPC / constructs, 2 ml of supernatant from overnight cultures with 10% (v / v) TCA (trichloroacetic acid) were precipitated on ice for 4 h, pelleted by centrifugation and in SDS- Sample buffer resuspended. 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 which is directed against the hemolysin portion of the fusion protein, tested for expression and secretion. C1 stands for the huZPC-1 construct (see Fig. 9), C2 stands for the huZPC-2 construct (see Fig. 9), C3 stands for the huZPC-3 construct (see Fig. 9) and pMO stands for for vector control.

Fig. 5 shows a Western blot in which various pMOhly1 / (m) ZPB / constructs were tested for expression and secretion. After transformation of E. coli DH5α with the various pMOhly1 / (m) ZPB / constructs, 2 ml of supernatant from overnight cultures with 10% (v / v) TCA (trichloroacetic acid) was precipitated on ice for 4 h, pelleted by centrifugation and in SDS- Sample buffer resuspended. 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 which is directed against the hemolysin portion of the fusion protein, tested for expression and secretion. B1 stands for the mZPB-1 construct (see Fig. 10), B2 stands for the mZPB-2 construct (see Fig. 10), B3 stands for the mZPB-3 construct (see Fig. 10), B4 stands for for the mZPB-4 construct (see Fig. 10) and pMO stands for the vector control.

Fig. 6 shows a Western blot in which various pMOMyl (hu) ZPB / constructs were tested for expression and secretion. After transformation of S. typhimurium SL7207 with the various pMO / (hu) ZPB / constructs, 2 ml of supernatant from overnight cultures with 10% (v / v) TCA (trichloroacetic acid) was precipitated on ice for 4 h, pelleted by centrifugation and in SDS- Sample buffer resuspended. 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 which is directed against the hemolysin portion of the fusion protein, tested for expression and secretion. B1 stands for the huZPB-1 construct (see Fig. 8), B2 stands for the huZPB-2 construct (see Fig. 8), B3 stands for the huZPB-3 construct (see Fig. 8), B4 stands for for the huZPB-4 construct (see Fig. 8), B5 stands for the huZPB-5 construct (see Fig. 8) and pMO stands for vector control.

Fig. 7 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. 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.

Fig. 8 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. 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.

Fig. 9 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 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 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.

Fig. 10 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 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 mZPB-1, huZPB-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 / Hly A fusion proteins. Finally, it is indicated whether secretion of the fusion proteins was detected with an antibody directed against the HlyA portion.

Bs it is believed that the person skilled in the art based on the present invention Description can run in its entirety. The following example is therefore used only the closer description and shows that the invention is feasible and should not in any Restrictive ways.

example Cloning and expression of huZPA, huZPB, huZPC and mZPB fragments in E. coli and S. typhimurium

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. Starting from mRNA isolated from ovaries of superovulating mice (Uwe Eberspächer, Schering AG), the corresponding cDNA was synthesized as follows: approx. 5 µg RNA were mixed in 32 µl DEPC-H ₂ O with 3 µl oligo-dT primer and added for 5 min Incubated at 65 ° C. The mixture is allowed to cool for 10 min at room temperature and the following reagents are added: 5 µl synthesis buffer, 5 µl 0.1 M DTT, 1 µl RNase inhibitor, 3 µl 25 mM dNTPs and 1 µl MMLV reverse transcriptase (20 U / µl) ( 1st Strand Synthesis Kit, Stratagene). This is followed by an incubation for 1 h at 37 ° C.

With the help of specific oligonucleotides (Table I), PCR-amplified gene fragments overlapping the corresponding cDNA were amplified, which have 5 'and 3' Nsi I restriction sites ( Fig. 7 to 10: Maps of the ZP genes with details of the primer positions and the resulting ones resulting gene fragments). The PCR amplification (Salki et al., 1988) was performed in a Thermocycler 60/2 (bio-med, Theres, Germany). For this, cDNA was amplified with the corresponding 5 'and 3' primers (1 µM final concentration each), dNTPs (200 µM each) and 2.5 U Tag DNA polymerase (Promega) and the manufacturer's buffer in a volume of 100 µl. Before the first cycle, 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. These PCR products were first inserted "bluntly" into the Sma I site of the vector pUC18. The ZP fragment was then cut out by an Nsi I digest and inserted into the expression vector pMOhly1 ( FIG. 1). After transformation of E. coli DH5α with the various pMOhly1 / ZP constructs, 2 ml of supernatant from overnight cultures with 10% (v / v) TCA was precipitated on ice for 4 h, pelleted by centrifugation and resuspended in SDS sample buffer. The supernatant proteins were separated by SDS-PAGE in a 15% polyacrylamide gel and, after transfer to nitrocellulose, tested for the secretion of zona / hemolysin fusion proteins. The expression and secretion of two huZPA fragments (A1 and A4, Fig. 2) and four huZPB fragments (B1-B4, Fig. 3) were detected using a polyclonal antibody directed against the hemolysin portion of the fusion protein . A transport-competent domain was identified for huZPC (C1, Fig. 4). Similar to huZPB, the secretion of three protein fragments of mZPB by the transport system could also be shown (B1-B3, Fig. 5).

The pMOhly1 derivatives described were then transformed into the strain S. typhimurium LB5000, which lacks the restriction system, so that E. coli DNA can be introduced efficiently into this strain. After isolation of the recombinant expression vectors from S. typhimurium LB5000, the strain S. typhimurium SL7207 was successfully transformed. As already described several times, this strain is suitable as a vaccination strain. The ZP / Hiy fusion proteins are also secreted in this strain. Fig. 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.

Claims (4)

1. Use of a secretion vector in the

• (a) are inserted from genes or gene fragments suitable for fertility control which (b) contain the complete hemolysin operon including the hly-specific promoter and an enhancer-like regulator hlyR and in which (c) a large part of the hlyA gene has been deleted, whereby
• (d) the proteins or protein fragments suitable for fertility control are synthesized and
• (e) by the secretion of these antigens by attenuated Salmonella or other Gram negative attenuated vaccine strains,
• (e) oral vaccination is generated.

2. Use according to claim 1, wherein the genes suitable for fertility control or Coding gene fragments for zona pellucida proteins. 3. Use according to claim 1, wherein the secretion vector is pMOhly1. 4. Use according to claim 1, wherein the Salmonella strain is Salmonella typhimurim.