Note: Descriptions are shown in the official language in which they were submitted.
CA 02288602 1999-11-08
WO 98/-50067 PCT/EP98/02679
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
CA 02288602 1999-11-08
2
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.
CA 02288602 1999-11-08
3
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.
CA 02288602 1999-11-08
4
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
CA 02288602 1999-11-08
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
CA 02288602 1999-11-08
6
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
CA 02288602 1999-11-08
7
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
CA 02288602 1999-11-08
8
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
CA 02288602 1999-11-08
9
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
CA 02288602 1999-11-08
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
CA 02288602 1999-11-08
11
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.
CA 02288602 1999-11-08
12
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
CA 02288602 1999-11-08
13
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
CA 02288602 1999-11-08
14
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
CA 02288602 1999-11-08
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.
CA 02288602 1999-11-08
16
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.
CA 02288602 1999-11-08
17
WO 98/50067 PCT/EP98/02679
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
CA 02288602 1999-11-08
18
~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
CA 02288602 1999-11-08
19
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.
CA 02288602 1999-11-08
WO 98/50067 PCT/EP98/02679
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
CA 02288602 1999-11-08
21
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.
CA 02288602 1999-11-08
22
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.
CA 02288602 1999-11-08
23
WO 98/50067 PCT/EP98/02679
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'
CA 02288602 1999-11-08
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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
