Nucleic acids encoding type I interferon variants

Variants of type I interferons containing peptide extensions, and their production using recombinant DNA techniques, are described. Expression cassettes comprising DNA coding for the variant interferon, DNA coding for a signal peptide, and a promoter are described for use in transforming yeast and in the production of the variant interferons.

BACKGROUND OF THE INVENTION 
The present invention relates to new variants derived from type I 
interferons, obtained from yeast by recombinant DNA techniques. 
Interferons were originally identified by their capacity for preventing 
viral replication. They constitute a family of proteins which have been 
divided into different groups. The classification currently proposed for 
interferons distinguishes two main types: 
type I which groups together .alpha., .omega. and .beta. interferons and 
trophoblastins; 
and type II which comprises .gamma. interferons. .alpha. and .omega. 
interferons were previously known under the respective names .alpha.-I 
interferon (class I .alpha.) and .alpha.-II interferon (class II .alpha.), 
and trophoblastins could have been classified among the .alpha.-III 
interferons. 
Apart from their antiviral activity, interferons can possess other 
functions. For example, it has already been established that, in some 
mammals such as cattle and sheep, trophoblastin participates in the 
phenomenon of maternal recognition of gestation. 
Ovine trophoblastin or oTP has been demonstrated, respectively, by MARTAL 
et al. [J. Reprod. Fert., 56, 63-73 (1979); Pro. 10th intern. Congress on 
Anim. Reprod. and A.1., Urbana-Champaign (USA), 11, 509 (short 
communication) 1984)] and GODKIN et al. [J. Reprod. Fert., 65, 141-150 
(1982)] in sheep, where it is produced in abundance by the embryo between 
the 12th and the 21st day of gestation; in cattle, it is produced between 
the 16th and the 24th day. It exists in at least 5 isoforms corresponding 
to different alleles. These isofoms are described in the international PCT 
application published under No. WO 89/08,706 in the name of the INSTITUT 
NATIONAL DE LA RECHERCHE AGRONOMIQUE. The molecular weight of 
trophoblastin is 20 kDa and its isoelectric point is between 5.3 and 5.5, 
depending on the isoform in question. Trophoblastins have been best 
characterized in certain ruminants. However, recent studies show that 
trophoblastin-like molecules also appear to exist in other mammals (horse, 
rabbit) and in man. 
The trophoblastin produced by embryos possesses like the other interferons, 
miscellaneous biological activities. 
Trophoblastin participates, in particular, in the mechanism of recognition 
of the embryo by the maternal body. 
In most cases, the length of a gestation period greatly exceeds that of the 
luteal phase of the ovarian cycle. When fertilization has taken place, 
certain mechanisms occur in order to prolong the life of the corpus luteum 
and to prevent a return of the ovarian cycle. In ruminants, the embryo 
emits a biochemical signal in the form of trophoblastin (oTP). This 
substance enables the body to continue to secrete progesterone, a hormone 
which is essential eryonic for normal embryonic development. 
GODKIN et al. [J. Reprod. Fert., 71, 57-64 (1984)] have shown that the 
intrauterine injection of purified oTP prolongs the secretion of luteal 
progesterone for a few days in recipient cycling ewes. FINCHER et al. [J. 
Reprod. Fert., 76, 425-433 (1986)] have found that the injection of 
natural oTP into the uterus delays oxytocin- or estradiol-induced 
luteolysis by several days and is accompanied by a reduction in 
prostaglandin F.sub.2.alpha. secretion. Similar observations have been 
made with recombinant class I .alpha. interferons [PLANTE et al., 
Endocrinology 122, 2342-2344, (1988); STEWART et al., (1989) J. Repr. 
Fert. Supp. p. 127-138]. 
Trophoblastin is secreted by the embryo only during a relatively short 
period: from the 16th to the 24th day of gestation as regards cattle and 
from the 12th to the 22nd day of gestation in the case of sheep. 
If the mother and the embryo are asynchronous, that is to say if the embryo 
secretes trophoblastin before the mother is physiologically capable of 
being sensitive thereto and of responding to such a stimulation, the 
corpus luteum regresses and the embryo dies. This possibility is 
especially critical when embryo transfer is being undertaken. To date, the 
failure rate in embryo transfer is greater than 30%. This is especially 
disastrous from an economic standpoint, the situation being still more 
marked in the case of in vitro fertilization. 
A large number of embryonic mortalities are considered to be due to the 
fact that the state of development of the embryo and the maternal 
physiological conditions do not appear to be "in phase" at the time of 
implantation of the embryo in the uterus of the carrying mother. In 
addition, embryos are often frozen before transfer, resulting in the loss 
of some capacity for production of trophoblastin produced by the embryos. 
An antiviral activity of trophoblastin has also been demonstrated, for 
example by MARTAL et al. [J. Reprod. Fert. Abs. series, 2, 3, (1988)] and 
PONTZER et al., [Biochem. Biophys. Res. Com., 152, 801-807, (1988)], as 
well as in the abovementioned PCT Application 89/08,706. This application 
also proposes the use of trophoblastin for preventing the rejection of 
organ transplants. 
These properties enable a large number of therapeutic uses of trophoblastin 
to be envisaged. The development of such applications has, however, come 
up against the fact that trophoblastin could be obtained only from concept 
uses, which meant that it could not be produced in sufficient quantities 
for in vivo use. 
In view of the foregoing, it would hence be very advantageous to have large 
quantities of trophoblastin available, in order to be able to treat 
animals at the start of gestation. Only recombinant DNA techniques can 
enable this objective to be achieved. 
The cloning in E. coli of the complementary DNA (cDNA) of the messenger RNA 
(mRNA) coding for ovine trophoblastin has been described in PCT 
Application 89/08,706, mentioned above. The construction, from this cDNA, 
of chimeric genes enabling trophoblastin to be produced in microorganisms 
could hence be envisaged. 
Nevertheless, the first attempts at expression of a cDNA coding for ovine 
trophoblastin proved inconclusive. These first attempts employed bacteria 
(E. coli) or yeasts (S. cerevisiae). In particular, an expression cassette 
intended for production of ovine trophoblastin in yeast, and comprising a 
DNA fragment coding for a signal peptide linked to the 5' end of the cDNA 
coding for mature ovine trophoblastin, did not enable synthesis to be 
obtained at an adequate level. 
Moreover, ZSEBO et al., [J. Biol. Chem. 261 (13): 5858, (1986)] report that 
an .alpha. interferon cannot be secreted under good conditions and in 
large quantities using the "prepro" system of the .alpha. factor. 
In addition, generally speaking, type I interferons are characterized by 
the presence of four cysteine residues at positions 1, 29, 99 and 139 
which link with one another to form the disulfide bridges cysl-cys99 and 
cys29-cys139, and it is considered that the cysteine residue at the 
N-terminal position is essential for the maintenance of a correct 
conformational structure. 
Surprisingly, the inventors have now found the addition of a DNA fragment 
coding for an additional amino acid, or for a di- or tripeptide such as, 
for example, for the dipeptide Ala-Pro or Ala-Gly, at the 5' end of the 
cDNA of an interferon possessing a cysteine at the N-terminal position to 
be beneficial. In effect, the whole of the coding structure is adequately 
expressed in yeast, and the variants thereby produced are secreted in 
larger quantities than those encoded by a structure lacking this fragment. 
Furthermore, it could be established that the variants obtained in this 
manner retain a biological activity similar to that of natural 
interferons. For example, the addition of a dipeptide at the N-terminal 
end of ovine trophoblastin gives rise to variants which retain the 
antiviral, immunological and antiluteolytic activities of natural 
trophoblastin. 
SUMMARY OF THE INVENTION 
Consequently, the invention proposes new variants of type I interferon, 
corresponding to one of the following formulae: 
EQU X.sub.1 --R.sub.0 (I) 
or alternatively 
EQU X.sub.1 --X.sub.2 --R.sub.0 (II) 
or alternatively 
EQU X.sub.1 --X.sub.2 --X.sub.3 --R.sub.0 (III) 
in which X.sub.1, X.sub.2 and X.sub.3 are identical or different and each 
represent an amino acid, 
and R.sub.0 represents the amino acid sequence of the mature form of a type 
I interferon. 
Preferably, X.sub.1, X.sub.2 and X.sub.3 each represent an acidic amino 
acid, a basic amino acid or an amino acid chosen from the group consisting 
of alanine, valine, proline, glycine, serine, threonine, cysteine, 
asparagine and glutamine, 
and X.sub.1 represents an amino acid other than proline. 
Type I interferon is understood, in particular, to mean an .alpha. 
interferon (IFN-.alpha.I) or .omega. interferon (IFN-.alpha.II) or a 
trophoblastin. An .alpha. interferon is characterized by a sequence of 166 
amino acids, while an .omega. interferon possesses a C-terminal extension 
of 6 amino acids. 
For a given species, the interferons generally exhibit some degree of 
natural allelic variety. For example, as regards the .alpha. interferons 
of human origin, at least about 15 genes are known, the coding sequences 
of which exhibit more than an 85% homology with one another. 
Advantageously, a variant according to the invention corresponds to one of 
the formulae: 
EQU Ala-Pro-R.sub.0 ' (IV) 
or 
EQU Ala-Gly-R.sub.0 ' (V) 
in which R.sub.0 'represents the amino acid sequence of the mature form of 
an .omega. interferon or of a trophoblastin. Preferably, a variant 
according to the invention corresponds to one of the formulae: 
EQU Ala-Pro-R.sub.0 " (VI) 
or 
EQU Ala-Gly-R.sub.0 " (VII) 
in which R.sub.0 " represents the amino acid sequence of the mature form of 
an .omega. interferon or of a trophoblastin of bovine or ovine origin. 
As an absolute preference, a variant according to the invention is of 
formula: 
EQU Ala-Pro-R.sub.0 '" (VIII) 
or 
EQU Ala-Gly-R.sub.0 '" (IX) 
in which R.sub.0 '" represents the amino acid sequence of the mature form 
of any one of the isoforms of ovine trophoblastin. 
Isoforms of ovine trophoblastin designated T1, T2, T3, T4 and T5, 
respectively, are described in PCT Application 89/08,706. 
In the context of the present invention, the preferred variants of formula 
(VIII) or (IX) include variants in which R.sub.0 '" represents the amino 
acid sequence of the mature form of any one of the isoforms T1 to T5 of 
ovine trophoblastin. 
FIG. 1 gives, as an example, the amino acid sequence of isoforms of 
trophoblastin, beginning with the cysteine residue at position 1 and 
ending with the proline residue at position 172 (signal peptide from -23 
to -1), in which: 
R.sub.5 is a glutamic acid, glutamine or arginine residue, 
R.sub.6 is an arginine or lysine residue, 
R.sub.35 is a lysine or aspartic acid residue, 
R.sub.44 is a glutamic acid or aspartic acid residue, 
R.sub.48 is a leucine or aspartic acid residue, and 
R.sub.49 is a leucine or glutamine residue. 
Preferred variants falling within the definition of the formulae (VIII) and 
(IX) include: 
a variant of formula (Xa) in which R.sub.5 is an arginine residue, R.sub.6 
is a lysine residue, R.sub.35 is an aspartic acid residue, R.sub.44 is a 
glutamic acid residue, R.sub.48 is a leucine residue and R.sub.49 a 
glutamine residue, 
a variant of formula (Xb) in which R.sub.5 is a glutamic acid residue, 
R.sub.6 is an arginine residue, R.sub.35 is a lysine residue, R.sub.44 is 
a glutamic acid residue, R.sub.48 is an aspartic acid residue and R.sub.49 
a leucine residue, 
a variant of formula (Xc) in which R.sub.5 is a glutamine residue, R.sub.6 
is an arginine residue, R.sub.35 is an aspartic acid residue and R.sub.44 
is an aspartic acid residue, and 
a variant of formula (Xd) in which R.sub.5 is a glutamine residue, R.sub.6 
is an arginine residue, R.sub.35 is an aspartic acid residue, R.sub.44 is 
a glutamic acid residue, R.sub.48 is a leucine residue and R.sub.49 a 
glutamine residue. 
Moreover, the subject of the invention is also a cassette for the 
expression of a variant according to the invention, which comprises at 
least: 
a first DNA fragment coding for a variant according to the invention, 
a second DNA fragment coding for a signal peptide, said second DNA fragment 
being linked to the 5' end of the first DNA fragment, 
a promoter enabling said DNA fragments to be expressed in yeast. 
Such a cassette is capable of promoting the expression of a peptide 
precursor consisting of a signal peptide on the N-terminal side, and a 
variant according to the invention on the C-terminal side. During passage 
through the endoplasmic reticulum, the signal peptide will be removed by 
cleavage to release the variant in mature form. 
For use in an expression cassette according to the invention, said second 
DNA fragment is such that it can code for any signal peptide whose 
C-terminal end constitutes a proteolysis site capable of being recognized 
by a signal peptidase of the host organism which is to harbor the 
expression cassette. The signal peptidase must necessarily cut the 
C-terminal end of the signal peptide. 
In the context of the present invention, advantageous signal peptides 
include, for example: 
the signal peptide of the precursor of the .alpha. factor having the amino 
acid sequence 
Met-Arg-Phe-Pro-Ser-Ile-Phe-Thr-Ala-Val-Leu-Phe-Ala-Ala-Ser-Ser-Ala-Leu-Al 
a (the proteolysis site is underlined); 
the signal peptide of the precursor of yeast .beta.-1,3-glucanase having 
the amino acid sequence 
Met-Arg-Phe-Ser-Thr-Thr-Leu-Ala-Thr-Ala-Ala-Thr-Ala-Leu-Phe-Phe-Thr-Ala-Se 
r-Gln-Val-Ser-Ala (the proteolysis site is underlined); as well as the 
functional derivatives of these peptides. 
For example, functional derivatives of the signal peptide of the precursor 
of .beta.-1,3-glucanase are described in the PCT application File No. 
FR90/00,306 of 27.04.90. 
Generally speaking, the promoter intended for the expression of the 
precursor of a variant according to the invention can be any promoter 
which is functional in yeast, preferably a promoter capable of inducing a 
good level of expression of any coding sequence. An advantageous promoter 
is, for example, the promoter of the MF.alpha.1 gene which codes for the 
.alpha. factor, or a functional derivative of this promoter. 
Lastly, the subject of the invention is also: 
a yeast cell transformed with an expression cassette according to the 
invention, and 
a method for producing a variant according to the invention, which 
comprises the act of culturing a yeast cell transformed with an expression 
cassette according to the invention and of harvesting said variant from 
the culture supernatant. 
The expression cassette according to the invention, as present in the 
transformed yeast cell, may be either incorporated in the genome of the 
yeast or carried by a plasmid capable of replicating in the yeast. In the 
latter case, it is appropriate to choose a plasmid/yeast host system such 
that the plasmid can be maintained in the yeast by selection pressure. An 
advantageous choice is of, on the one hand a yeast host that is 
auxotrophic for a metabolite which is essential to cell growth, and on the 
other hand a plasmid which enables this auxotrophy to be complemented. 
The interferon variants according to the invention may be used in all the 
applications of natural interferons, such as, for example, the production 
of antiviral, immunomodulatory, anti-inflammatory and antitumor 
medicaments. They may also be used as immunogens for inducing the 
production of anti-type I interferon antibodies. 
In addition, the trophoblastin variants obtained according to the invention 
(these variants are hereinafter designated by the general term APrT) are, 
apart from the general applications of type I interferons mentioned above, 
used more specifically for the production of antiluteolytic medicaments as 
well as of products intended for improving the survival of embryos when 
they are transplanted, and for the production of reagents permitting 
diagnosis of the viability of embryos at an early stage of their 
development. 
According to a preferred embodiment of the present invention, APrT is used 
for the treatment of herds and flocks, in order to improve their 
fertility. 
According to another preferred embodiment of the present invention, APrT is 
used for the treatment of embryos when they are transplanted, in the 
various techniques of reproduction of breeding animals involving embryo 
transfer, especially those associated with cryo-preservation, with in 
vitro fertilization, with embryo cloning and with embryo transgenesis. 
According to yet another preferred embodiment of the present invention, 
APrT is used for the production of reagents and kits permitting the 
diagnosis of viability of embryos at an early stage of their development. 
Such a diagnosis is based on the assay of the trophoblastin produced by the 
embryos. This assay can be, for example, performed by immunological 
methods; in this case, APrT may be used as an immunogen for the production 
of antibodies, or as an antigen, in competitive type methods. The 
trophoblastin produced by embryos may also be quantified by its antiviral 
activity; APrT may be used as a standard of antiviral activity in such an 
assay. 
The immunological properties of APrT may also be turned to good account in 
order to induce the appearance of anti-trophoblastin antibodies in an 
animal in which it is desired to produce infertility. 
The subject of the present invention is also a method for purifying APrT 
from culture medium of yeasts that produce it, in which method the APrT is 
purified by chromatography (for example of the DEAE type) on an anion 
exchange column with a three-step elution: 
a KCl gradient from 0 to 0.135M 
an isocratic phase at approximately 0.135M KCl 
a KCl gradient from 0.135M to 0.5M, the APrT being collected at between 
0.135 and 0.3M KCl. 
APrT may also be purified by reverse-phase chromatography, or by affinity 
chromatography, using anti-trophoblastin antibodies.

DETAILED DESCRIPTION 
A better understanding of the invention will be gained from the further 
description which follows, which relates to examples of preparation of 
variants of type I interferon according to the invention. 
It is, however, self-evident that these examples are given only by way of 
illustration of the subject of the invention and in no way constitute a 
limitation thereof. 
EXAMPLE 1 
Construction of the plasmid for expression of the Ala-Pro variant of ovine 
trophoblastin in yeast (pTG7908) 
The DNA sequence coding for the precursor of ovine trophoblastin, the 
latter being as described in PCT Application WO 89/08,706 (see also FIG. 
1), is cloned in the form of an EcoRI fragment into the vector M13TG131 
described in the paper by M. P. KIENY et al., Gene (1983) 26:91. The 
vector M13TG771 is thereby obtained. In order to be able to isolate the 
fragment coding for the mature protein, that is to say the DNA fragment 
without the signal sequence, a HindIII site is created at the 5' end of 
the mature sequence of the protein by directed mutagensis, using the 
AMERSHAM kit and the oligonucleotide OTG2102, the sequence of which is as 
follows: GAGGATCTCAAGCTTGTTACCTAT. 
The antisense strand of trophoblastin, carried by the single-stranded 
vector M13TG771, is represented below in line I, and the oligonucleotide 
OTG2102 is represented in line II; the stars (*) represent the mismatches 
which will cause the desired mutations. 
##STR1## 
The vector M13TG7720 is thereby obtained. The point mutations which have 
been introduced into the coding sequence induce the replacement of the 
amino acids leucine at position -2 and glycine at position -1 of the 
precursor of trophoblastin by glutamine and alanine, respectively. 
M13TG7720 is then digested with EcoRI, and the EcoRI DNA fragment coding 
for the mutated precursor of trophoblastin is inserted into the vector 
pTG769 (described in Patent Application EP 0,258,118) digested beforehand 
with EcoRI. The vector pTG7901 is thereby obtained. 
Moreover, in order to produce trophoblastin in yeast, the DNA fragment 
coding for trophoblastin must be placed under the control of a yeast 
promoter. For this purpose, the vector M13TG3841 is used, this vector 
being described in the PCT patent application filed on Apr. 28, 1990, the 
file number of which is FR90/00,306, and containing, in particular: 
the promoter of the gene coding for the alpha 1 factor (MFalpha1); signal 
peptide of the precursor of the alpha 1 factor; and 
the pro sequence of MFalpha1. 
An SmaI restriction site is created between the second and the third codon 
of the pro sequence of MFalpha1 by directed mutagenesis, using the 
Amersham kit and oligonucleotide OTG2072 whose sequence is as follows: 
TCCGCATTAGCTGCTCCCGGGAACACTACAACAGAA. 
The antisense strand of the prepro sequences of MFalpha1, carried by the 
vector M13TG3841, is represented below in line I, and the oligonucleotide 
OTG2072 is represented in line II; the stars (*) indicate the mismatches 
which will cause the desired mutations. 
##STR2## 
Vector M13TG3869 is thereby obtained. The point mutations which have been 
introduced into the coding sequence induce the replacement of glycine by 
valine. 
The vector pTG7901 is digested with HindIII to liberate the HindIII DNA 
fragment coding for mature trophoblastin, which is then treated with mung 
bean nuclease. This fragment is inserted into the vector M13TG3869 
digested beforehand with SmaI. The vector M13TG7740 is thereby obtained, 
which vector contains, in sequence and in frame: 
the MFalpha1 promoter, 
the pre sequence of MFalpha1, followed by the first two codons of the pro 
sequence, that is to say those coding for the amino acids alanine and 
proline, and 
the DNA fragment coding for ovine trophoblastin. 
The SphI DNA fragment derived from the vector M13TG7740, containing the 
MFalpha1 promoter, the pre sequence followed by the alanine and proline 
codons and the DNA sequence coding for mature trophoblastin, is inserted 
into the yeast vector pTG3828 (described in the PCT patent application the 
file number of which is FR90/00,306) digested beforehand with SphI. 
Plasmid pTG7908 is thereby obtained. 
By performing directed mutagenesis of the vector M13TG7740, using the 
Amersham kit as described above and the oligonucleotide OTG2643, 
replacement of the sequence coding for the N-terminal extension Ala-Pro by 
a sequence coding for Ala-Gly is obtained. An SphI fragment of the vector 
M13TG7745 obtained in this manner is inserted, as described above, into 
plasmid pTG3828; the resulting plasmid is designated pTG7941. 
A plasmid designated pTG7904, lacking the sequence coding for the dipeptide 
Ala-Pro, was also constructed by directed mutagenesis of M13TG7740, by 
means of the oligonucleotide OTG2299 and insertion of an SphI restriction 
fragment into pTG3828. FIGS. 2A and 2B summarize the protocol for 
obtaining pTG7908, pTG7904 and pTG7941. 
EXAMPLE 2 
Production of the variant Ala-Pro-trophoblastin by yeast 
A yeast strain of the species Saccharomyces cerevisiae, of genotype 
MATalpha, ura3-251,-373,-328, leu2-3,-112,his3,pep4-3, is transformed with 
plasmid pTG7908 by the lithium acetate method [H. ITO et al., J. 
Bacteriol. (1983) 153], and the uracil prototrophs (Ura.sup.+) are 
selected on a YNBG medium (14 g/l of Yeast Nitrogen Base, 10 g/l of 
glucose) with the addition of 10 g/l of casamino acids. 
To compare the level of expression of the different variants encoded by the 
plasmids described in Example 1, clones of cells transformed with the 
plasmids were cultured in flasks at 30.degree. C. to an OD.sub.600 of 8 to 
10 units, in order to determine the production of trophoblastin in the 
culture supernatants. 
The production of recombinant trophoblasin by yeasts transformed, 
respectively, with plasmids pTG 7904, pTG 7908 and pTG 7941 was evaluated 
by acrylamide gel electrophoresis, staining with Coomassie blue and 
comparison with a standard protein preparation (PHARMACIA). 
The results are illustrated in the following table. 
TABLE I 
______________________________________ 
Recombinant trophoblastin 
Plasmid (mg/l/OD.sub.600) 
______________________________________ 
pTG 7904 0.06 to 0.25 
pTG 7908 2 to 2.5 
pTG 7941 1 to 1.5 
______________________________________ 
These results show that the presence of the N-terminal extension of 2 amino 
acids brings about a substantial increase (from 4- to 10-fold) in the 
production of recombinant trophoblastin by yeast. 
The variant Ala-Pro-trophoblastin is produced by "Fed-Batch" in a 
BIOLAFFITE 20-L fermenter. 12 l of Kappeli medium D [A. FICCHTER et al., 
Adv. Microbial. Physiol. (1981), 22, 123-183], concentrated 1.5-fold and 
containing 10 g/l of glucose and HY Case SF (sold by SHEFFIELD), are 
inoculated with 400 ml of a preculture of a yeast clone transformed with 
pTG9708. This preculture is prepared in an Erlenmeyer at 30.degree. C. on 
a YNBG selective medium. Before fermentation is started up, the OD.sub.600 
of the medium thus inoculated is 0.2. Fermentation is carried out at 
30.degree. C., at a pH of 4.5 controlled by the addition of 10% ammonia 
solution, and at a partial pressure of oxygen corresponding to 30% of the 
saturation pressure, held constant by regulation of the agitation. When 
all the glucose has been consumed, the OD.sub.600 is measured (OD of start 
of feeding), and feeding with glucose in exponential steps of 3 hours is 
begun, knowing that the specific growth rate (.mu.) is 0.1 hour.sup.-1 and 
that the quantity of glucose added (QS) is 0.045 g/h/OD. Fermentation is 
stopped when ODe.sub.600 =100, and harvesting is carried out by 
centrifugation at 5000 g. 13 l of culture supernatant containing large 
quantities of the variant Ala-Pro-trophoblastin are thereby obtained. 
EXAMPLE 3 
Purification of the variant Ala-Pro-trophoblastin from culture supernatants 
On a semi-preparative scale 
The yeast culture supernatants are centrifuged, then concentrated and 
dialyzed against 0.05M Tris-HCl buffer (pH 8.2) in an ultrafiltration cell 
(AMICON) across a FILTRON membrane that retains molecules above 10 kDa. 
Isolation of the APrT was performed on a semi-preparative scale by high 
performance liquid chromatography (HPLC) on a semi-preparative TSK 
DEAE-5PW anion exchanger column (150.times.21.5 mm) equilibrated with 
0.05M Tris-HCl buffer pH 8.2. The flow rate is 4 ml/min. After injection 
of the sample, elution is performed with a KCl gradient from 0 to 0.135M 
(0.05M Tris-HCl buffer pH 8.2) for 90 min, followed by an isocratic 
plateau phase at 0.135M KCl for 30 min and then a second gradient up to 
0.5M KCl for 80 min. The elution is monitored by measuring the absorption 
at 280 nm. The peak corresponding to APrT is identified using an 
anti(natural trophoblastin) immune serum (the similarity of the 
immunological properties of APrT and natural trophoblastin is demonstrated 
below in Example 4), and the corresponding fractions are then collected. 
The APrT emerges at the end of the isocratic plateau phase, at between 
0.135M and 0.25M KCl. 
FIG. 3(a) illustrates the elution profile obtained; the peak corresponding 
to APrT is shaded. 
The apparent molecular weight of the APrT in polyacrylamide gel 
electrophoresis in the presence of SDS is approximately 21 kDa. FIG. 4 
shows the electrophoretic profile obtained (well a: APrT; well b: 
molecular weight markers). 
Preparative purification 
The purification is performed on a preparative TSK DEAE-5PW column 
(200.times.55 mm). Elution is performed with a KCl gradient in 0.05M 
Tris-HCl buffer pH 8.3 at a flow rate of 25 ml/min, under the following 
conditions: 
gradient from 0 to 0.135M KCl for 80 min; 
isocratic phase at 0.135M for 40 min; 
gradient from 0.135 to 0.5M KCl for 120 min. 
The elution profile is shown in FIG. 3(b). The shaded peak corresponds to 
APrT. 
EXAMPLE 4 
Comparison of the immunological properties of APrT with those of natural 
trophoblastin 
The existence of immunological cross-reactions between APrT and 
trophoblastin is evaluated by radio-immunoassay (RIA), using an 
anti-trophoblastin polyclonal antiserum and an iodine-125-labeled 
trophoblastin preparation, according to the protocol described in PCT 
Application 89/08,706. 
The RIA inhibition curves obtained under these conditions are shown in FIG. 
5; the Log of the dilution is plotted as abscissa, and the corresponding 
Logit=Log (Bo/1-Bo) as ordinate (Bo represents the maximal concentration 
of bound trophoblastin for a constant concentration of anti-trophoblastin 
antiserum). 
(.cndot.) control: purified preparation of natural trophoblastin; 
Y=-0.9572X-0.2341 
linear regression coefficient=0.9957 
(x) culture medium of yeasts secreting APrT (dilutions from 1 to 1/100); 
Y=-0.9372X -0.921 
linear regression coefficient=0.9996 
(*) APrT preparation (dilutions from 1 to 1/4000) obtained as described in 
Example 1; 
Y=-0.9341X-2.41 
linear regression coefficient=0.9955 
These curves are parallel to one another, thereby demonstrating that the 
immunological properties of APrT are similar to those of natural 
trophoblastin. 
EXAMPLE 5 
Test of antiviral activity 
The antiviral activity of APrT is measured according to the protocol 
described by LA BONNARDIERE and LAUDE [Infection and Immunity, 32, 28-31, 
(1981)] on MDBK (Madin Darby Bovine Kidney) cells, a calf kidney cell line 
in the presence of vesicular stomatitis virus. 
The results obtained are compared with those obtained using a reference 
interferon, which is a porcine .alpha. interferon having a titer of 1,000 
IU, itself calibrated with respect to the human reference standard. 
The results of this test of antiviral activity reveal an equivalent 
activity for the yeast culture medium supernatants (0.8.times.10.sup.8 
IU/mg), purified APrT (0.55.times.10.sup.8 IU/mg) and natural 
trophoblastin (0.7.times.10.sup.8 IU/mg) (the quantity of trophoblastin is 
determined by radioimmunoassay). 
EXAMPLE 6 
IN vivo demonstration of the antiluteolytic activity of APrT 
Animals and hormone treatment 
The experiment was carried out on 30 Prealpes-du-Sud breed ewes. The estrus 
cycles are synchronized by means of vaginal sponges impregnated with 300 
mg of 17.alpha.-acetoxy-9.alpha.-fluoro-11.beta.-hydroxyprogesterone 
(SEARLE, INTERVET). These sponges are left in place for 14 days and, on 
the day of withdrawal (D14), the ewes are injected intramuscularly with 
500 IU of PMSG (pregnant mare serum gonadotrophin); 48 hours later, the 
ewes begin a new cycle (D0). 
Insertion of intrauterine catheters 
The ewes are anesthetized and then, by laparotomy at the linea alba, the 
operator gains access to the uterus and marks the corpora lutea using 
Indian ink. A sterile catheter (SILASTIC, DOW CORNING) 0.076 mm in 
internal diameter, 0.165 mm in external diameter and 70 cm in length is 
equipped with a short sleeve at one of its ends, which enables the 
catheter to be held in place in the uterine horn, substantially at the 
uterotubal junction. The opening is closed with a needle crimped to a 
catgut thread (Laboratoire BRUNEAU). A purse-string suture is performed 
around the insertion orifice of the catheter, effecting durable 
positioning of the device. A safety stitch made with a needle crimped to 
silk (Laboratoire BRUNEAU) attaches the catheter to the broad ligament. 
The other end is closed with a flax thread, and a loop is made so that the 
operator, after piercing the abdominal wall on the right-hand side, can 
take out the catheter by pulling the loop. A SILASTIC check ring was 
placed 30 cm from the intrauterine end in order to limit the projection of 
the catheter and to act as a stop inside the abdominal wall. A length of 
approximately 40 cm is at the disposal of the operator who performs the 
intrauterine injections A stitch made around the abdominal emergence 
orifice of the catheter enhances the robustness of the device. The 
insertions of catheters are performed between the 9th and 11th days of the 
cycle. 
Intrauterine infections 
Three groups of ewes were formed: the intrauterine injections begin between 
the 10th and the 12th day of the cycle, and are performed twice a day over 
8 days. The APrT is dissolved in physiological saline containing 50,000 
IU/ml of penecillin G and 0.2% BSA (bovine serum albumin). The volume of 
solution injected is 1 ml. 
Group A: This is a control group composed of 10 ewes which receive, twice a 
day, 1 ml of 0.2% BSA (fraction V, SIGMA) solution in physiological saline 
(0.9% NaCl) containing 50,000 IU/ml of penecillin G. 
Group B: This group is composed of 8 ewes to which 170 .mu.g of APrT have 
been administered twice daily. 
Group C: This group is composed of 4 ewes which receive 80 .mu.g of APrT 
twice a day. 
Group D: 5 ewes receive 340 .mu.g of APrT twice a day. 
At the end of the experiment, all the ewes undergo an exploratory 
laparotomy in order, on the one hand to verify that the catheters have in 
fact stayed in place, and on the other hand to monitor in each group the 
presence or absence of corpus luteum (or corpora lutea) marked with Indian 
ink. 
Radioimmunoassay of progesterone 
Blood of the animals is drawn from the jugular vein using Vacutainer tubes 
(BECTON-DICKINSON) without anticoagulant. The serum progesterone 
concentration is determined by direct radioimmunoassay without extraction, 
according to the protocol described by HEYMAN et al. [J. Reprod. Fert., 
70, 533-540, (1984)]. Tritium-labeled progesterone and a specific 
anti-progesterone immune serum (PASTEUR INSTITUTE) are used for this 
assay. 
In the control group A, the progesterone concentration in the peripheral 
blood decreases abruptly in all the ewes from the 14th day, reaching 
levels below 0.5 ng/ml between the 15th and the 17th day post-estrus. The 
mean duration of the cycle in this group is 15.2.+-.0.3 days. The 
administration of 80 .mu.g of APrT per day (group C) does not prolong this 
duration. 
In group B (170 .mu.g/day), a slowing down of the fall in the blood 
progesterone level compared to group A is seen at the 14th day of the 
cycle: 7 ewes out of 8 exhibit at this stage progesterone levels above 1 
ng/ml (against 4 out of 10 in group A); at the 15th day of the cycle, 5 
ewes out of 8 still exhibit progesterone levels above 1 ng/ml (against 2 
out of 10 in group A). 
In this group, luteolysis is delayed on average by 2 days compared to group 
A. 
In group D, the intrauterine administration of APrT at a dose of 340 
.mu.g/day maintains luteal function well beyond the duration of the normal 
cycle in four ewes out of 5 (25, 32, 45 and 64 days, respectively, in ewes 
nos. 9037, 9431, 9458 and 9053). 
The comparative mean profile of progeserone secretion between the different 
groups, shown in FIG. 6, shows clearly that there is a marked persistence 
of luteal activity in group D. 
() Group A 
() Group B 
(*) Group C 
(.quadrature.) Group D 
In addition, during the surgical monitoring by laparotomy, no newly formed 
corpus luteumwas seen in any of the 4 ewes of group D which are mentioned 
above, showing that the measured blood progesterone level corresponds to 
the persistence of the cyclic corpora lutea which preceded the injections 
of APrT. 
Possible side effects of APrT were looked for: the mean temperature of the 
ewes of the test groups was taken daily. 
No difference was observed between the temperature of the control animals 
of group A and that of the animals of the other groups. 
More generally speaking, no behavioral disturbance (loss of appetite, and 
the like) was observed in the animals treated with APrT, compared to the 
control animals. The same applies to the blood picture (red cells, white 
cells, platelets) and to the serum transaminase (SGPT) levels. 
Intramuscular injections 
The same experiment was performed, injecting APrT intramuscularly. 
One group of ewes (E) received 2 injections of APrT solution daily (2 mg of 
APrT/day: 1 mg in the morning, 1 mg at night). 
A group of control ewes (F) received injections of a BSA solution (2 
mg/day: 1 mg in the morning, 1 mg at night). 
The persistence of luteal activity in the animals of group E is similar to 
that observed in the animals of group D (treated by intrauterine 
injection). 
The only side effect observed is a slight rise in the mean temperature of 
the animals of group E compared to those of group F. In contrast, no 
behavioral modification is observed, and neither are modifications of the 
blood picture and the serum transaminase (SGPT) levels. 
Experiments carried out previously showed the antiluteolytic activity of 
natural trophoblastin, but they did not permit the assertion that 
trophoblastin sufficed on its own to prevent luteolysis, and had not 
enabled the role of the different isoforms of trophoblastin to be 
determined. Now, the experiments described show that the APrT obtained 
from a single isoform suffices, at suitable doses, to inhibit luteolysis, 
despite the two additional amino acids of the N-terminal end. Lastly, no 
apparent sign of proline toxicity is observed. 
EXAMPLE 7 
Demonstration of the immunosuppressant properties of APrT 
These properties were demonstrated by four types of tests enabling 
different modes of action to be demonstrated: 
antimitotic activity, evaluated by the action on the proliferation of 
mouse, human or sheep lymphocytes; 
inhibitory activity with respect to the cytolytic graft rejection reaction, 
evaluated by means of the in vitro test of the mixed lymphocyte reaction 
(MLR); 
inhibitory activity with respect to the in vivo local graft rejection 
reaction (local graft versus host reaction); 
immunoregulatory activity with respect to the population of NK killer 
lymphocytes, which are independent of the antigens of the major 
histocompatibility complex (MHC). 
1) Action of APrT on the proliferation of mouse lymphocytes activated with 
phytohemagglutinin 
Mouse lymphocytes are obtained from C3H/He or Balb/c mouse spleen after 
blending in a Potter and washing twice in RPMI 1640 culture medium at 1500 
rpm for 10 min. Finally, the isolated lymphocytes are mixed in the same 
culture medium, to which 10% of fetal calf serum (FCS) is added, at a 
final concentration of 5.times.10.sup.6 cells/ml. The culture medium is 
composed of 500 ml of RPMI 1640 (GIBCO)+5 ml of penicillin G/streptomycin 
(GIBCO)+5 ml of 7.5% sodium bicarbonate (GIBCO)+5 ml of glutamine. 
100 .mu.l per well of culture medium containing 6.times.10.sup.5 
lymphocytes, activated with 5 g/ml of phytohemagglutinin (PHA) (WELLCOME), 
are incubated with 100 .mu.l of an APrT solution at a concentration of 3 
.mu.g/ml (.perspectiveto.10.sup.8 IU/mg), or 100 .mu.l of culture medium 
(control), in 96-well microtest plates at 37.degree. C. in an air/CO.sub.2 
(95%/5%) atmosphere for 48 h. 
Lymphocyte proliferation is evaluated by measuring the incorporation of 
tritiated thymidine. 25 .mu.l of [.sup.3 H] thymidine (0.04 mCi/ml) are 
added to each well, and the cells are harvested 24 h later and deposited 
on filters (Glass Microfibre Filters-GFM-WHATMAN). After drying, the 
filters are placed in tubes to which 1 ml of scintillation fluid 
(ECONFLUOR) is added. The radioactivity is measured in a .beta.-radiation 
counter (BECKMAN). 
The results are illustrated in FIG. 7, which shows that APrT inhibits very 
markedly (55%) the proliferation of mouse lymphocytes treated with PHA. 
A: Control 
B: APrT 
The radioactivity in cpm is plotted as ordinate. In the presence of human 
or sheep lymphocytes activated with phytohemagglutinin A (PHA), APrT 
likewise inhibits lymphocyte replication. This inhibition does not result 
from a cytotoxic effect of APrT, since cell viability is not affected by 
APrT, which is shown by incubating the lymphocytes in the presence of 
trypan blue or of .sup.51 Cr. 
2) Action of APrT on a mixed lymphocyte reaction 
The mixed lymphocyte reactions are carried out by incubating, per well, 150 
.mu.l of culture medium containing 5.times.10.sup.8 C3H/He responding 
cells per ml with 5.times.10.sup.6 isogeneic or allogeneic stimulator 
cells irradiated at 1,800 rads/ml. 
C3H/He mouse cells are used for the isogeneic reaction and Balb/c mouse 
cells for the allogeneic reaction. 100 .mu.l of APrT at a concentration of 
3 .mu.g/ml (10.sup.8 IU/mg), or of culture medium (control), is added per 
well in 96-well microtest plates (FALCON 3072) at 37.degree. C., and the 
cultures are left under an air/CO.sub.2 (95%/5%) gaseous atmosphere for 4 
days. 
Lymphocyte lysis by the cytotoxic lymphocytes produced during the mixed 
lymphocyte reaction is evaluated by measuring the incorporation of 
tritiated thymidine. 25 .mu.l of [.sup.3 H] thymidine are added 24 h 
before sampling lymphocytes and depositing them on filters. Measurement of 
the radioactivity of the filters is performed in a scintillation counter. 
The results are shown in FIG. 8: 
A: Control 
B: Natural trophoblastin 
C: APrT 
The radioactivity in cpm is plotted as ordinate. 
In two-way culture of lymphocytes originating from two mouse strains 
(Balb/c and C3H/He), APrT inhibits to the extent of 90% the lysis of mouse 
lympho-cytes by the cytotoxic cells (CTL) produced. 
3) Local graft rejection reaction 
APrT in the proportion of 2 .mu.g/ml is added to a suspension of allogeneic 
spleen cells originating from Balb/c mice, which is injected into the 
plantar pads of a hind foot of F.sub.1 (Balb/b.times.B.sub.6) recipient 
mice. 
The other hind foot, used as a control, is injected with the spleen cells, 
but without APrT. The popliteal lymphocytic ganglia are removed 4 to 6 
days later (depending on the group of mice) and weighed. Cells of these 
ganglia are removed and activated with PHA; the incorporation of [.sup.3 
H] thymidine in these cells is measured. 
The results are shown in FIGS. 9(a) and 9(b), which show that the weight of 
the lymphatic ganglia in the cells treated with APrT is decreased compared 
to the control cells (FIG. 9a), and that the incorporation of tritiated 
thymidine in the cells originating from the popliteal ganglia of the feet 
treated with APrT is also lower than in the control cells (FIG. 9b. 
These results show that APrT inhibits in vivo the graft rejection reaction, 
even in a species (mouse) very distant from the ovine species. 
4) Action of APrT on cell lysis by NK cells 
K562 cells (human erythroleukemic line) are centrifuged for 10 min at 1,800 
rpm, and 0.5 ml of .sup.51 Cr is deposited on the pellet. After 1 h of 
incubation at 37.degree. C. in 5% CO.sub.2 /95% air, the cells are washed 
three times in RPMI culture medium and resuspended in the same culture 
medium at a concentration of 2.times.10.sup.5 cells/ml. 
Three types of incubation in microtitration plates at 37.degree. C. in an 
air/CO.sub.2 (95%/5%) atmosphere are carried out in parallel: 
100 .mu.l of labeled K562 cells+100 .mu.l of human lymphocytes 50 to 100 
times more concentrated+100 .mu.l of APrT at a concentration of 3 .mu.g/ml 
(10.sup.8 IU/mg), or of culture medium (control), enable the radioactive 
proteins of the experimental medium (exp. rad. prot.) to be determined; 
100 .mu.l of labeled K562 cells+200 .mu.l of 4N HCl enable the radioactive 
proteins of the total medium (tot. rad. prot.) to be evaluated; 
100 .mu.l of labeled K562 cells+200 .mu.l of culture medium enable the 
radioactive proteins of the natural medium (nat. rad. prot.) to be 
determined. 
After 4 h of incubation, 100 .mu.l of supernatant are sampled from each 
well, and the radioactivity due to the release of .sup.51 Cr-labeled 
proteins into the medium is counted in cpm in a gamma-radiation counter. 
The results are expressed as the mean percentage cell lysis of samples, and 
calculated according to the following formula: 
##EQU1## 
FIG. 10 shows the results obtained: 
A: Control 
B: APrT 
C: IFN-alpha 
D: IFN-gamma 
It is hence clearly apparent that APrT activates the cell lysis of K562 
target cells by NK cells. 
This activation of cell lysis by NK cells is similar to that observed using 
class I human .alpha. interferon, and less than that of the reference 
human .gamma. interferon. 
As is apparent from the foregoing, the invention is in no way limited to 
those modes of implementation, embodiments and modes of application which 
have just been described more explicitly; it covers, on the contrary, all 
variants which may occur to the specialist in the field, without departing 
from the scope or compass of the present invention.