FR2696188A1 - Feline CD4 protein and corresp. DNA - for study of feline immunodeficiency virus and for therapy - Google Patents

Abstract

The invention relates to the cat CD4 molecule and its applications, in particular to the constitution of a veterinary medicinal product.

Description

CAT4 MOLECULE, NUCLEIC SEQUENCE ENCODING FOR
THIS MOLECULE AND THEIR APPLICATIONS8
The invention relates to a new molecule having the characteristic properties of the cat CD4 molecule. It also relates to the products which can be derived therefrom, as well as to the applications which can be made thereof for the diagnosis of an immunodeficiency syndrome acquired in cats, or even its prevention or its treatment. Finally, this molecule opens perspectives on the possibility of creating models making it possible to test the efficacy of active principles of drugs against acquired immunodeficiency syndromes induced by retroviruses of the same types in other species, even human AIDS.

 The CD4 molecule, already identified in a number of species, is expressed primarily in cells of the immune system. It plays a critical role in the differentiation and activation of T lymphocytes. It also plays a role in the development cycle of the immunodeficiency virus acquired in humans HIV (1). The human CD4 molecule constitutes an expression product of a gene belonging to the super-family of immunoglobulin genes. It has an extracellular segment successively comprising four domains of the immunoglobulin type respectively called D1-D4, a transmembrane region and a cytoplasmic tail (2).

The human CD4 molecule participates both in adhesion reactions between cells and in signal transduction (1,7). It has been suggested that
CD4 interacted with class II molecules of the major histocompatibility complex (MHC: abbreviation of the English expression "Major
Histocompatibility complex "), but this has not been fully demonstrated on a bio-physical level (8,9).

 Besides its role in the immune system, the CD4 molecule is the HIV receptor (1). The HIV envelope glycoprotein has a strong affinity for and binds to the D1 region of the human CD4 molecule. It results from analyzes carried out on the effects of mutations induced in CD4 in the context of the crystallographic structure of its D1-D2 regions, a small region of 10 acids would constitute the main, if not unique, site for binding of the gp120 glycoprotein. HIV on human CD4 (10). This loop is located on a face opposite to that of the CD4 region presumed to interact with NHC class II molecules.

 This invention between the HIV envelope glycoprotein and the CD4 molecule is essential for the viral cycle of the retrovirus. It is considered responsible for the characteristic tropism of primate lentiviruses, such as HIV-1, HIV-2 or SIV (11>.

 CD4 molecules have also been identified in mice and rats. They have structural similarities with the human CD4 molecule (3,4). Therefore, it has been envisaged to use such animals as possible models of human infection with HIV. But such a project ran up against the fact that the cells of these animals cannot be infected by the human retrovirus. the same was true for transgenic mice, the cells of which had been modified by incorporating into their genomes a gene sequence coding for the human CD4 molecule under conditions allowing their expression.

 A number of retrovirus strains capable of inducing acquired immunodeficiency in cats (IVF): (abbreviation of the English expression "Feline Immunodeficiency Virus") have been recently isolated (12). Whereas in vivo, like HIV; IVF leads to a progressive depletion of CD4 lymphocytes, the selective tropism for these cells seems to be controversial; indeed, some strains show selective tropism for CD4- lymphocytes (13). On the other hand, prior incubation with a monoclonal antibody against cat CD4 (14) does not or very little inhibit infection of a culture of cat CD4 + lymphocytes by IVF. To date, the existence of an interaction between the already isolated IVF strains and the cat CD4 molecule has still not been demonstrated.

 The cloning of the cat CD4 molecule is essential for the analysis of the tropism of the various IVF isolates, and can improve our knowledge regarding the acquisition of the tropism for the CD4 molecule by lentiviruses.

The invention relates more particularly to this cat CD4 molecule and to the nucleic sequence coding for this molecule. The cloning strategy (see Figure 1) of this molecule was based on the assumption that some of its regions must have a high sequence homology with corresponding regions of other CD4 molecules already described in the literature, among others those of humans, mice and rats. Now, it is known that regions of strong homology exist in the cytoplasmic part of these CD4 molecules and that regions of weaker homology also exist in the extra-membrane part of these CD4; in particular, in the D3 domain of these 3 molecules, there is a well-preserved region comprising in each case a glycosylation site. The hypothesis has therefore been formulated that degenerate nucleic primers capable of hybridizing to the corresponding regions of the molecules
Human and mouse CD4 could possibly also hybridize to corresponding regions of the cat's CD4 molecule. This hypothesis has proved fruitful, insofar as the implementation of an amplification technique by polymerization chain reaction (PCR, according to the abbreviation of the English expression "Polymerase
Chain Reaction ") provided access to the mRNAs encoding the
Cat CD4 and, through them, to the molecule
Cat CD4.

 The first primer ("primer-sense") coding for the amino acid sequence: AGSGNLTL was modeled on the highly conserved V3 region of the amino acid sequence of the CD4 molecule of humans, mice (L3T4) and rat (W3 / 25) and the second primer ("antisense primer") coding for the amino acid sequence EKKTCQC corresponding to the cytoplasmic region of these 3 molecules (see Figure 1A).

 The oligonucleotides were synthesized by an oligonucleotide synthesizer (Pharmacia).

The 5 'degenerate oligonucleotide (CAT / J3) corresponding to the "primer-sense" consisted of a degenerate 27-mer comprising 256 nucleotide variants all coding for the amino acid sequence AGSGNLTL. Its sequence can be represented by the following formula 5'TTGCTGG (T / C / G / A) TCTGG (T / C / G / A) AACCT (T / C / G / A) AC (T / C / G / A )
CTGG3 '.

 The 3 'oligonucleotide (CYT / R) was a degenerate, antisense 30-mer, also comprising 256 variants of nucleic acids all encoding EKKTCQC, and had at its 3' end an EcoR1 restriction site. The overall sequence of this 30-mer can be represented by the following general formula 5'GGGAATTCGA (G / A) AAtG / A) AA (G / A) AC (T / C / G / A) TG (C / T) CA (G / A) TG (C / T) C3 '.

To allow rapid amplification of the ends of the cDNA according to the method described by
Frohman et al. (15) a hybrid primer (hereinafter called "adapter-dT17" has been synthesized comprising an oligo (dT) (17 residues) conjugated to a primer (adapter) formed of an oligonucleotide also synthesized for this purpose and comprising 17 bases 5'GACTCGAGTCGACATCGA 3 'The sequence of the "adapter-dT17" is then the following 5' GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTTTT 3 '.

 These degenerate oligonucleotides were used to amplify a cDNA obtained from cat lymphatic nodules. This first step (Figure 1B) was to lead to the isolation and amplification of a major band comprising approximately 450 base pairs. After cloning and sequencing of this fragment, it appeared that the amino acid sequence which could be deduced therefrom exhibited characters of homology with the corresponding regions of the human and murine CD4 molecules, while significantly differing therefrom.

In order to clone the remaining part of the molecule
Feline CD4 by amplification, the RACE procedure (15) (Figure 1B) was implemented. This would involve amplification between a specific primer and either a non-specific primer hybridizing with the natural end "3"'or with a synthetic 5' end (poly A). In this way two fragments could be cloned, one comprising the 3 'end and including the above-mentioned sequence of 450 base pairs, and the other comprising the 5' end and also including the sequence of 450 pairs basis of the feline CD4 molecule. From these two fragments, the cDNA of the nucleic sequence coding for the feline CD4 molecule (3,2) kilo-bases) could be reconstituted. The complete sequence of these clones could be determined by displacement (walking) along the cloned DNA sequence, using specific sequential synthetic primers.

 The nucleic acid sequence of the cDNA coding for the feline CD4 molecule according to the invention is shown in Figure 2A.

 The amino acid sequence (shown in accordance with the code for the unique letter designating amino acids) is shown in Figure 2B.

It also appears in FIG. 3 in which the corresponding sequences of the human and murine CD4 molecules are also indicated, after alignment of their respective identical amino acids, to account for the relative homologies between these molecules and the feline CD4 molecule. In addition to these relative homologies, the molecule
Cat CD4 has a unique characteristic characterized by the presence of an insertion of 17 amino acids, rich in serine / threonine and located at the beginning of the D2 domain (amino acids 108 to 123).

 1) The invention therefore relates more particularly to the cat CD4 protein, responding to the amino acid sequence of FIG. 1 or any peptide fragments derived from this protein, since they contain the binding site for IVF. In particular, the invention relates to the fragment of the soluble feline protein CD4 +, which comprises the regions D1 and D2 (respectively extending from the amino acid numbered 1 to amino acid 101, and from amino acid 102 to 1 amino acid 196 of FIG. 3) which appear in FIG. 2B, or even the only region D1. These fragments of polypeptides can be obtained from the whole protein by cleavage thereof by enzymes (for example achromase, trypsin, alpha-chymotrysin, anti-xa factor, etc.) which are also capable of recognizing particular peptide bonds and selecting those of the fragments capable of interfering with an IVF virus, preselected as will be indicated below, in tests for infection of CD4 + cells with this IVF virus. As a variant, the fragments of the above-mentioned polypeptides can be obtained by expression of corresponding nucleic acid sequences derived from a cDNA coding for the entire cat CD4 molecule after insertion of these sequences into an appropriate recombinant vector and transformation of appropriate cells into which these sequences can then be expressed.

2) The invention therefore also relates to veterinary pharmaceutical compositions containing the above-mentioned protein or the corresponding polypeptide, in combination with a pharmaceutically acceptable excipient. Preferably, the composition is injectable. In the latter case, it is in particular in the form of an aqueous, sterile solution, preferably isotonic. It goes without saying that these compositions are then dosed so as to ensure the effectiveness of the active ingredient in terms of prevention or inhibition in vivo of possible infection of the CD4 lymphocytes of the feline host by virus variants IVF likely to interact with its CD4 molecules. This molecule may contain all or part of the feline protein CD4, soluble, containing the D1 and D4 regions (from amino acid 1 to amino acid 385), which appear in the Figure 2B and capable of interfering with an IVF virus. The fragment comprising these four regions could have been expressed by Chinese hamster ovary (CHO) cells. For this effect, the gene coding for these regions (from amino acid 1 to amino acid 385) was inserted into a plasmid containing the gene coding for dihydrofolate reductase (DHFR) (Figure 4A). This plasmid was then introduced into CHO cells
DHFR negative. The introduction of this gene first allows the selection of cells producing the protein of interest, then the amplification of the production of this protein by the use of a structural analogue of the natural substrate of this enzyme ( 16). Under these conditions, the molecule produced by these cells binds in a specific way to the monoclonal antibody described as recognizing the CD4 molecule of cat (14), when a technique of interaction receptor-ligand in real time is used (17 ) (see Figure 5). If necessary, the cat CD4 molecule (or part of this CD4> is covalently conjugated to an antivisual principle, for example a ricin A chain, an active region of the exotoxin A of Pseudomonas, or to any other agent anti-viral, immunomodulator capable of interfering with the pathogenic effect of these IVFs. The cat's CD4 molecule (or active part thereof) can also coexist with the above-mentioned antiviral principle or analog within a protein recombinant (see for example the articles by Mark A. Till et al, Science, Vol. 242 (1988), pp. 1166-1168 and Vijay K. Chaudhary et al,
Nature, Vol. 335 (1988), pp. 369-372 which describe techniques of this type, but applied to the molecule
Human CD4). It is then observed that the CD4 molecule contributes to the targeting of the antiviral principle towards infected T lymphocytes of the host.

 3) The invention also relates to recombinant vectors comprising a nucleic sequence coding for said feline CD4 protein, whether or not deleted from the insertion of 17 amino acids or for the corresponding active fragments, under the control of a promoter recognized by cellular hosts. susceptible to be transformed by such recombinant vectors.

4) The invention relates more particularly to cell cultures initially CD4 transformed with the above-mentioned recombinant vector and therefore made CD4 +. Of particular interest are such cells, especially when they can be grown as attached cells and when they can be infected with IVF strains previously adapted to these cells. For this purpose, the gene coding for the entire molecule (domains D1 to
D4, transmembrane and cytoplasmic regions) of cat CD4 was inserted into an expression vector (Figure 4B); this plasmid was then introduced into 3 types of cell line: CrFK, Fc3Tg and CHO. These CD4 lines were in these conditions made CD4 + (see Figure 6). The implementation of such transformed cells can then allow the selection of those of IVF isolators which will be shown the ability to interact with an active site of the CD4 molecule of cat. These transformed cells can then be used to study the degree of interference with the
IVF selects drug substances under study, when tests of in vitro infection of cultures of these cells are carried out with the IVF thus adapted, in the presence of the drug principle studied. The degree of activity of the drug principle studied is then a function of the degree of inhibition of the infectivity of the IVF used relative to that measured in controls.

5) The invention also relates to the use of such transformed lines (with or without the insertion of 17 aa) for the purpose of selecting or adapting one or more IVF isolate (s). The tropism of IVF for the cat CD4 molecule is disputed and has not yet been clearly demonstrated; the hypothesis that the ancestral form of IVF had a specific tropism for this molecule and then gradually lost it over time is advanced. On the other hand, the presence of the insertion of 17aa could have contributed to the loss of this tropism, hence the interest in transforming cell lines without this insertion. it would therefore be interesting to select or adapt one or more isolates having "re-acquired" a specific tropism for this molecule and therefore being capable of infecting CD4 + cells. The method would consist in continuously adding high concentrations of
IVF in the culture medium of adherent CD4 + cells (with or without insertion) in order to isolate an infectious strain.

6) The invention also relates to the use of mouse or hamster cells expressing the molecule
Feline CD4 with or without insertion and analysis of their susceptibility to infection by IVF or by a previously selected or adapted isolate (see 5)) or by an HIV / IVF hybrid (see 7)) for the purpose to develop transgenic animals.

 7) The invention also relates to the use of an HIV / IVF hybrid having all the HIV sequences but where the gene coding for the envelope of HIV would have been replaced by that coding for the envelope of an isolate from IVF having a specific tropism for the CD4 feline molecule. This isolate could be a "natural" isolate, that is to say selected from the IVF isolates already described in the literature, or else an "adapted" isolate resulting from an adaptation (see 5)).

 8) The invention also relates to the use of the insertion of 17 aa into another CD4 molecule, in particular that of humans, in order to observe the influence that it would have on HIV infection for example.

 9) The invention also relates to the use of all or part of the cat CD4 molecule in laboratory tests (ELISA, BIAcore, etc.) involving its interaction with the envelope glycoprotein of FIV variants capable to infect CD4 + cells.

 10) Finally, the invention relates to the development of transgenic mice whose genetic heritage has been modified by incorporating into the genomes of their cells a recombinant sequence containing a sequence coding for the cat CD4 molecule (with or without the insertion) under the control of a promoter more specifically active in lymphoid cells. These transgenic animals (for example obtained by a method equivalent to that described in European patent No. 85304490.7 filed on June 24, 1985 with the EPO) can then become, in turn, excellent animal models for the study of infection with IVF, in particular by an adapted or selected isolate or by an HIV / IVF hybrid. They can also be used to study the drug effects of a substance under study with regard to active immunodeficiency of cats, and even humans.

The legends of figures 1 to 6 are indicated
v below.

Figure 1: Strategy for cloning cat CD4 cDNA
Fig. 1A: A fragment of cat CD4 cDNA (CD4 cDNA in the drawing) was obtained by an amplification technique using two degenerate primers derived from two highly conserved regions between the amino acid sequences of human CD4 molecules ( T4), mouse (L3T4) and rat (W3 / 25).

The first primer, sense for AGSGNLTL is localized in the V3 region and the second, antisense for EKKTCQC is localized in the cytoplasmic region.

Fig. 1B: Cloning of the entire cDNA of cat CD4 by the RACE technique.

Figure 2
Sequences of nucleic acids (A) and amino acids (B) of the cat CD4 molecule.

Figure 3: Alignment of the amino acid sequences of
CDI molecules of man (H), of mouse
(S) and cat (C)
The numbers on the right represent the position of the amino acids. The Dl, D2, D3 and D4 domains and the transmembrane (TM) and cytoplasmic (CYT) regions are indicated by horizontal arrows. The N-glycosylation sites appear in shaded form. The identical amino acid homologies between humans, mice and cats for each domain are as follows: D1, 44%; D2, 32%; D3, 50%; D4, 44%; TM, 39% and CYT, 70%.

Figure 4: Expression vectors of the gene coding for
the cat CDO molecule
Fig. 4A: The inserted gene codes for the extramembrane part of cat CD4 leading to the production of the feline CD4 molecule in a soluble recombinant form.

Fig. 4B: The gene inserted into the vector codes for all of the cat's CD4, thus allowing its membrane expression in a given cell line.

Figure 5: Demonstration of the soluble feline CD4 recombinant protein (fsT4). Interaction of the culture supernatant of transfected CHO cells with the plasmid containing the gene coding for sfT4 with a cat anti-CD4 monoclonal antibody (A) or an irrelevant monoclonal antibody (B). (resonance limits (RU) as a function of time t (in seconds).

FIG. 6A: Analysis by flow cytometry of the expression of the cat CD4 molecule on the surface of CHO cells using the anti-cat CD4 monoclonal antibody (Fel7) (FIG. 6B) and by way of comparison using a monoclonal antibody that does not react with the cat CD4 molecule.

Figure 6B: comparison as a control of a non-cross-reactive antibody.

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Hendrickson and R. P. Sekaly. Mutational analysis of the interaction between CD4 and class Il MHO: class Il antigens contact CD4 on a surface opposite the gpl20-binding site. Cell. 66: 1037. (1991).

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Claims (11)

 1. Cat CD4 molecule, the amino acid sequence of which is shown in FIG. 2 or active fragment of this molecule as soon as it contains the binding site of an IVF retrovirus capable of binding to the entire molecule.  2. Molecule according to claim 1, characterized in that it is coupled with an anti-viral or immunomodulatory molecule capable of interfering with the pathogenic action of an IVF.  3. Molecule according to claim 2, characterized in that it is constituted by a recombinant molecule.  4. DNA sequence coding for the molecule or active fragment of the molecule of claim 1.  5. DNA sequence according to claim 4, the nucleotide sequence of which corresponds to that of FIG. 2.  6. DNA sequence according to claim 4 or 5, characterized in that it is fused with a sequence coding for the above-mentioned antiviral or immunomodulatory molecule.  7. Recombinant vector capable of transforming a determined cell host and containing a nucleic acid sequence according to any one of claims 4 to 6, placed under the control of a promoter recognized by the polymerases of this cell host, and this in conditions allowing its expression in this cellular host.  8. Recombinant vector according to claim 7, characterized in that it is capable of transforming cat or mouse lymphold cells and in that the promoter is specific to the corresponding lymphoid cells.  9. Cell host transformed by a recombinant vector according to claim 7 or claim 8.  10. A method of selecting IVFs interacting with a cat CD4 molecule characterized by bringing an infected cell culture supernatant into contact with a cell host according to claim 9 and by selecting those of the supernatant retroviruses which, in because of their affinity for CD4 expressed within this cellular host, is able to penetrate into it.  11. Veterinary pharmaceutical composition containing the CD4 molecule according to any one of claims 1 to 3 or a water-soluble part thereof.