AU645274B2

AU645274B2 - Method for the preparation of an immunizing composition

Info

Publication number: AU645274B2
Application number: AU65118/90A
Authority: AU
Inventors: Jean-Francois Bouquet; Yahia Chebloune; Francois-Loic Cosset; Antoine Drynda; Catherine Legras; Victor-Marc Nigon; Arielle Rey-Senelonge; Corinne Ronfort; Gerard Verdier
Original assignee: Institut National de la Recherche Agronomique INRA
Current assignee: Institut National de la Recherche Agronomique INRA
Priority date: 1989-09-29
Filing date: 1990-09-28
Publication date: 1994-01-13
Anticipated expiration: 2010-09-28
Also published as: CA2067172A1; IE903499A1; WO1991005055A1; JPH05500509A; AU6511890A; PT95469A; EP0420759A1; FR2663228A2

Description

The present invention relates to a process for the preparation of an immunizing composition using integration vectors arid expression vectors of a foreign gene in host cells.

According to the invention, an immunogenic protein which is produced on cells and s presented on membrane fragments of said cells is equipped with a considerably improved immunogenic capacity with respect to said purified protein.

More precisely, the present invention relates to a process for the preparation of an immunizing composition which is composed of the combination of an immunogenic protein and of cell membrane fragments, said protein being produced by said cells by presenting itself in an integrated form with their membranes.

According to a first embodiment of this invention, there is provided a process of preparing an immunizing composition consisting of the combination of an immunogenic protein and cell membrane fragments, wherein said immunogenic protein is produced by the expression in suitable host cells of a defective vector for the integration and expression of a foreign gene coding for said protein in said host clls, said protein being produced by said host cells and presented in a form integrated into their membranes.

According to a second embodiment of this invention, there is provided a process for preparing an immunizing composition against the RSV virus according to the first embodiment, characterized in that said protein is the enyprotein.

According to a third embodiment of this invention, there is provided a process for the preparation of an immunizing composition against Newcastle disease according to the first embodiment, characterized in that said protein is the heamagglutinin neuraminidase (HN) of the Newcastle virus.

According to a fourth embodiment of this invention, there is provided an 25 immunizing composition, obtained by the process of the first, second or third embodiments.

According to a very advantageous characteristic of the present invention, the immunogenic protein is produced by the expression, in suitable host cells, of an integration and expression vector of a gene encoding said protein in said host cells.

30 More particularly, said integration and expression vector will be a defective retroviral vector which contains a selection gene and the gene of said immunogenic protein, this vector being constructed in such a way as to optimize the production of the protein and so that the protein integrates itself into the membrane of the cells in which it is produced.

S The compositions according to the invention advantageously contain an adjuvant, in particular Freund's complete adjuvant.

The subject also of the present invention is a process for the preparation of a composition, characterized in that it comprises the following steps: IG:\WPUSER ULBVVO1014 9:TCW 1A a) the immunogenic protein is produced by expression, in appropriate host cells, of an integration and expression vector of a foreign gene encoding said protein in said host cells, the vector being constructed in such a way as to optimize the production of said protein and so that the protein integrates 5 1 a a a a a OR C o.

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go a GI:\WPUSER\ULBW00149:TCW 2 itself into the membrane of the cells in which it is produced, b) the host cells are subjected to a selection, in particular by the antibiotic which corresponds to the selection gene, and the cells which have thus been selected are multiplied, c) the resulting cells are treated in such a way as to inactivate the cell and viral nucleic acids which they contain, and d) the membrane constituents associated with the immunogenic protein are recovered and purified, if necessary.

In a particular embodiment of a process for the preparation of an immunizing composition according to the invention, the process comprises the following steps: production of a defective retroviral vector presented in the form of plasmid DNA containing a selection gene and the gene of an immunogenic protein, the vector being constructed in such a way as to optimize the production of the immunizing protein, the structure of this protein and of the integration vector being constructed in such a manner that the protein integrates itself into the membrane of the cell in which this protein is produced; transfection of this vector on a helper cell culture, resulting in the production of a viral preparation (helper-free) of this same vector in the form of a defective virus which is capable of causing infection; the resulting viral preparation is used for infecting a culture of ordinary cells so that no transmissible virus can be formed; the cells are subsequently, subjected to a selection by the antibiotic which corresponds to the selection gene used in a manner known by those skilled in the art; the cells' which have been selected in this manner o are multiplied using a traditional method; the resulting cells are treated in such a way as to 3 inactivate the cell and viral nucleic acids which they contain, in particular by UV irradiation followed by a freezing/thawing step, and the membrane constituents which are associated with the immunogenic protein are recovered, these constituents being purified, if necessary.

This preparation method has proved particularly successful and, to date, has never been used in the preparation of a vaccine.

US Patent 4,323,555 describes a preparation process for a composition which vaccinates against the BLV virus, which composition contains cell membrane fragments. However, these compositions are obtained from tumour cells which are infected by the entire BLV virus.

More precisely, these cells are derived from ovine and bovine lymphosarcomas caused by the BLV virus, and they can be used only for a vaccination against BLV. This process does not permit the preparation of a composition which contains a specific defined immunological protein which is associated with cell membrane fragments. As is the case in the process according to the invention which can be applied to the preparation of various different vaccine compositions as a function of the gene which is used as the antigen, introduced into the vectors and encoding a given protein. In effect, the process according to the invention makes it possible to choose immunization with the aid of a particular protein starting from its structural gene which is inserted into the vectors. It is also possible according to the invention to bring about expression of several antigens in these same cells, in order to obtain compositions which immunize against various viral infections.

Moreover, the process according to the invention does not utilize tumour cells and permits control of its various parameters. In effect, in the process according to the invention, the cells are infected in vitro by recombinant virions obtained Sfrom retrovira vectors which are defective as regards their multiplication. The step in which the recombinant virions f 4 are produced is controlled. The rec inant virions are produced on.helper cells. Since they are defective, these virions do not multiply on the cells which are later used as vaccination material. Moreover, the vector constructions according to the invention permit the production of a specific immunological protein to be optimized, as has been shown. Finally, the process according to the invention permits selection of the immunizing cells which express the antigen carried by the vectors. This selection is carried out in vitro starting from genes for antibiotic resistance which are carried by the vectors simultaneously with the gene(s) of interest for vaccination.

In a particular embodiment of the process according to the invention, avian vectors are used which are cultivated on avian cells, in particular CEF cells (chicken embryo fibroblasts) or LMH chicken liver cells.

An immunogenic protein which is produced on avian cells and presented on membrane fragments of said avian cells is equipped with immunogenic capacities which are considerably improved, even for mammals, compared with said purified protein.

The use of an avian retroviral vector for preparing such a protein permits perfect control of the process of integrating the gene for the immunogenic protein and the process of producing said protein in avian cells. In this way, all the factors can be optimized such as the transcription activity, the production of the protein and the presentation of this protein on receptors which are capable of multiplying its immunogenic activity. All these results are more difficult to obtain by an ordinary transfection step.

The use of an avian vector naturally necessitates avian cell culture. However, the same result may be obtained with vectors and cells of another origin, for example a mammal vector which is cultured on mammal host cells, such as a murine vector.

Viral integration and expression vectors for heterologous genes in avian cells have been described in Sthe European and international patent applications 5 EP 178,996 and PCT/FR 88 00487 (WO 89/03877). It would be advisable to refer to these publications for a better understanding of the present patent application and, more particularly, for the detailed description of avian viral vectors which can be used according to the present invention, in particular vectors pTXN3' and In a particular embodiment of the invention, there will be used, as a vector for the integration and the expression of the gene for the immunogenic protein in avian cells, a vector which is composed, completely or partly, of the proviral genome of avian erythroblastosis or of a related virus in which the gene for the immunogenic protein and the selection gene replace the genes v-erbA and v-erbB, and in that said genes are either under the control of an LTR promoter of the same virus, in which case said genes mime the genes which they replace, or under the control of a heterologous promoter, in which case an additional att sequence is positioned upstream of said heterologous promoter gene.

The avian viruses to which the present invention relates more particularly are AEV and the viruses of the ALSV type which have similar properties, as well as nondefective RAV type viruses.

In an embodiment which is particularly useful for the integration in CEF chicken cells or LMH liver cells, the gene for the immunogenic protein is under the influence of avian LTR, and their translation mechanism mimes that of the genes which they replace.

In a suitable manner, the vector contains the selection gene in the v-erbA position and the gene(s) for the immunogenic protein in the v-erbB po-ition.

According to another feature of these vectors, the selection gene and the gene for the immunogenic protein are advantageously translated from the AUG of the gag gene, of from their own AUG, but always in the same reading frame as that of the gag gene. If need be, the selection gene is therefore translated from the AUG of the gag gene and the gene for the immunogenic protein is fH^ translated from the AUG of the gag gene or its own AUS.

1 6 A stop codon is advantageously introduced between the gag gene and the gene for the immunogenic protein or the AUG beloining to said gene for the immunogenic protein.

The stop codon is preferably located at a distance of 60 to 70 nucleotides from the AUG codon of said gene for the immunogenic protein, in particular In another embodiment of the invention, the integration and expression vec&or is such that the selection gene is in the v-erbA position and dependent on an avian LTR5' promoter, 'aid, the gene for the immunogenic protein is in the '-.ybB position and dependent on a heterologous promoter, the vector now containing a supplementary att sequence positioned upstream of the heterologous promoter.

In vectors which have a supplementary att sequence, it is possible to delete part of the sequence of LTR5' in such a manner that, after a retroviral cycle, nothing but the supplementary att sequence which ensures integration remains functional.

It is preferred to carry out a deletion of 23 pb in the 3' terminal region of the U5 sequence of The vector is advantageously characterized in that the gene for the imunogenic protein is in the v-erbB position which iA framed by the heterologous promoter and the heterologous polyadenylation sequence, and in that the unit of promoter, gene for the immunogenic protein and polyadenylation sequence is in the same orientation, or in opposite orientation to the retroviral sense of transcription.

As a heterologous promoter, the promoter of the simian virus SV40 may be mentioned in particular.

To carry out the process according to the invention, it is likewise necessary to use permanent helper cell lines which are capable of producing helper-free viral preparations. The helper lines provide the defective genome with the proteins gag-pol-env which are necessary for it, permit the formation of virions and Stherefore permit the use of defective viral vectors without extra helper viruses.

I r 7 As a vector capable of transforming a normal avian cell into a helper cell, it will be possible to vitilize, according to the present invention, in particular a vector which contains all, or part, of the three genes gag-pol-env of the virus RAV-1 or the virus RAV-2, placed under the transcriptional control of an LTR of the same virus on which various deletions have been carried out to suppress the capacity of the RNA produced by this vector fo- eneapsidation.

In ae ambodiment, the starting cells used for preparing the helper cells are QT6 quail cells.

In one embodiment, in particular when the integration and expression vector consists, as the gene for the imlnunizing protein, of the env gene, it has been possible to suppress the env gene in the vector intended for the transformation of normal cells into helper cells. In other embodiments, the gag and pol genes can be deleted and replaced by a selection gene, the remaining env gene being preceded by its splicing acceptor site.

One example of an embodiment of the invention is an immunizing composition against the RSV virus, characterized in that said protein is the protein env associated with membrane fragments of CEF cells.

In another example, the subject of the present invention is an immunizing composition against Newcastle disease. More precisely, in one embodiment of the invention, the protein is haemagglutinin neuraminidase (HN) of the Newcastle virus.

As mentioned above, it will be found that other characteristics of avian vectors which can be used in the process according to the invention are described in the international patent application WO 89/03877.

Other characteristics and advantages of the present invention will appear in the light of the following detailed description of one embodiment.

Example 2 is illustrated by Figures 1 to Figure 1 represents the construction of the vector SK+Hn- 4 Figures 2 to 4 represent the constructions of the 8 vectors pHnN 1 pHnN Z and pHnN 3 Figure 5 represents the structure of the vectors of Figures 2 to 4 which are the vehicle for the Hn gene of the NDV.

Figure 6 represents the plasmid chart pNDV-71: ATG translation initiation codon TGA translation termination codon R Tcl R resistance genes, carried by the sequence pLBR322, to the antibiotics ampicillin and tetracycline; ori origin of replication of the plasmid F, L residue of the F and L genes of the NDV.

Hn Hn gene polyAF, poly AHn polyadenylation sequences of the messages of the F and Hn genes.

The big boxes represent the LTRs, the Hn gene and the selection gene. The small boxes represent residues of retroviral genes. The fine lines represent RNA transcribed from the vectors, while the bold lines represent the synthesized proteins. The white or black triangles represent the codons for translation initiation or termination, ds and as are splicing donor and acceptor sites.

EXAMPLE 1 Composition immunizing against the RSV virus which is responsible for avian leucosis A vector was employed which contains an env gene of the avian retrovirus RAV-1, belonging to sub-group A.

The animals (chickens) received this gene, or the products of its avtivity, in various forms which will be given in detail below. The animals which had been treated in this way were tested on the one hand for anti-env antibody prouction (titrated by seroneutralization of a viral preparation), and, on the other hand, for production of tumours after challenge resulting from the infection of an RSV.A virus (ROsus Sarcoma Virus, sub-group A) preparation (3.6 103 PFU/animal), 35 days after the first immunization treatment.

4 -9 1. Construction of the vectors The. env genes of sub-groups A and B, originating from RAV-1 and RAV-2, respectively, were inserted into the base vector pTXN5' (already carrying the neo gene), in which previously the sequence J (containing the AEV splicing acceptor site) and the residue of the env gene from AEV had bie deleted. These retroviral vectors called pNEA aid pNEB have been described in patent WO 89/03877 in E xple 2-b.4).

The non-ericapsldable vector pGPH, which serves for tr. jforming normal avian cells into helper cells, was prepared from pHFla, in which the hygro gene, which mtparts hygromycin B resistance, had been inserted in place of the env gene (downstream of the splicing acceptor site). This vector pGPH, which carries tha gag and pol genes from RAV-1, has been described in patent WO 89/03877, Example 4 5.1.4.

2. Obtaining the transcomplementing lines HAYDEE and

ISOLDE

QT6 quail cells were transfected by the DNA of the vector pGPH and subjected to hygromycin selection. 23 hygro' colonies were isolated and tested for extra- and intracellularproduction of p27 a 8 Half of these clones produced a quantity of p27 8 s which was equivalent to that produced by QT6 cells which had been infected by RAV-1.

9 of these lines were transfected by the DNA of the vector pNEA and subjected to neomycin selection. The supernatants of these cuAtures were titrated for production of particles which impart neoaycin resistance (in RFU/ml). From amongst these cultures, 2 produced particles in a significant amount under helper-free conditions, and one of these cultures produced a very high titre (more thai' 10i RFU/ml). The name HAYDEE was given to the hydrot clone which can produce this last-mentioned culture. This clone is therefore transcomplementing for the expression of a defective vector which carries an env gene.

The line HAYDEE was transfected by the DNA of the non-endapsidable vector pPhEA, which is described in patent 10 application WO 89/03877 in Example 4 5.1.6. This vector carries the-phleomycin resistance gene (phleo gene) as well as the env gene of sub-group A. After phleomycin selection, phleo' clones were isolated. 11 of these clones were tested for env gene production using an interference test, thanks to a superinfectant viral stock from sub-group A. In effect, the protein encoded by the env gene saturates the specific surface receptors of the sub-group and prevents reinfection by a retrovirus from the same sub-group. From amongst these clones, 2 proved to be as interfering as QT6 quail cells which had been infected by the virus RAV-1. 4 of these clones were transfected by the DNA of the defective retroviral vector pNL53, which is described in patent application WO 89/03877 in Example The supernatants of these cultures were titrated for their capacity to transmit the vector pNL53 to target celly under helper-free conditions. The most productive clone was given the name ISOLDE; it is capable of producing retroviral vectors which are entirely defective, at a titre of the order of 10 4 to 10 5 RFU/ml.

3. Properties of the NEA virus QT6 quail cells were infected by the retroviral vector pNEA produced by the line HAYDEE which had been transfected by pNEA. QT6 clones resulting from this infection were isolated (after neomycin selection). These clones were tested for the production of the env gene by an interference test with a viral stock which i~4-rferes in a pseudotype manner in sub-group A.

It turned out that 80 of these clones expressed the env gene, testifying to the capability of the vector NEA to transfer expression of the env gene to cultured cells.

4. Preparation of the immunizing material 4.1. Defective NEA virus (treatment a) in Table I) The NEA virus produced by the transcomplementing line HAYDEE which had been transfected by the vector pNEA was concentrated 100-fold by ultracentrifugation (30,000 rpm, 4°C, 20 minutes). The viral stocks are preserved at

C.

11 4.2. Supernatant of the line ISOLDE (treatment b) in Table I) This supernatant, which produces retroviral proteins with the exclusion f genetic retroviral material, was concentrated 100-fold (same conditions as for the NEA virus).

4.3 Preparations made with CEFs which had been infected by the vecto NEA (treatment c) in Table I) Chicken embryo fibroblasts (CEF) are infected by the helper-free viral preparation of the vector NEA, and then selected by G418. The neo cells are then amplified, and their supernatant is tested for the absence of the wild-type virus. These cells are irradiated by UV (at 254 nm) for 5 minutes, subjected to trypsin treatment, and then resuspended in PBS at a concentration of 3 x 106 cells per ml. The preparations are then deep-frozen at and thawed just before injection of the animal.

4.4. Preparations made with CEFs which had been infected by the RAV-1 virus (treatment d) in Table I) CEFs are infected by a supernatant of the RAV-1 virus and amplified. Before they are harvested using the same treatments as described above (in the presence of the RAV-1 virus is checked.

Injections of the animals The most complete results to date were obtained from animals which had an age of 3 months at the beginning of the treatment, and -"'ich were divided into groups of 9 to 11 animals which wt.-e given the same treatment (see Table I) 5.1. Protocol of the immunization The animals received one intravsnous injection using the treatments a) (approximately 10 7 RFU/enimal), b) (1 ml per animal), c) (3 x 1,0 recombinant cells per animal), d) (3 x 10 6 virem cells per animal), and e) (3 x 106 control cells per animal).

A booster injection is given 15 days after the S first injection, using the same methods.

J oT aN 9 12 A blood sample is taken every 8 days and used for preparing serum for testing for, the presence of antibodies.weeks after the first injection, all the animals (except those which had been treated using method d) are injected subcutaneously with 3.6 x 103 PFU/animal of a viral preparationof RSV of sub-group A.

5.2. Results 1) Checking for neutralizing antibodies Principle: The neutralizing antibodies specifically destroy the infectious activity of the retrovirus against which they are directed. To check the sera of treated animals, we used a viral stock NL/RAV 1 (vehicles for the Neo and Lac Z genes) which had been incubated with dilutions of the test sera 1/2, 1/4, 1/8192), which was then used for infecting QT6 cells on which B-galactosidase activity was detected by X-Gal coloration.

The results obtained by zeroneutralization with various dilutions of sera made it possible to define, for each serum, the dilution which allows neutralization of and 100 of the NL virus ND 5 s and NDio 0 13 TABLE II (4)

-I

(h) 1 I0 0 7 21 47 85 129 6000 <128 2 0 2 21 45 83 116 144 172 2000 <128 3 0 3 11 33 57 95 106 123 6000 64 8 30 47 83 155 216 NT NT 640 4 0 0 0 3 5 13 26 58 >8000 <256 6 0 0 1 4 9 33 101 211 4800 64 7 0 0 11 18 27 56 84 148 6000 <32 8 0 0 0 0 2 10 36 64 000 <128 9 0 3 11 23 54 83 118 147 4000 32 NP 300 300 NT NT NT NT NT NT sera tested; (2) dilution of sera number 1/16; (b) of B-gal cells per 1/64; 1/256; 1/512; 1024; 1/2048; 1/4096; (h) 1/8192; ND50; ND100; NP unprotected TABLE I: The results of the seroneutralization, represented by the number of blue cells per dilution of the serum employed. The last dilution which neutralizes 100 of the NL virus (ND 100) and the dilution which allows neutralization of 50 of the virus: ND 5 s are represented in the last two columns. The ND 50 values in the penultimate column are determined from curves established using the neutralization values obtained by the dilutions of each serum and given in columns 2 to 9. The sera of the 9 animals studied correspond to treatment c (see Table II), The result of an experiment is given in Table II below 14 TABLE II (7) a)NEA HF 9 0 9/9 0 100 0% b)ISOLDE 0 2/9 7/9 22 78 96 33 96 c)CEF NEA 9 9/9 0/9 100 %9 0% d)CEF RAVI 9 9/9 not 100 not not tested tested tested Control CEFs 10 0/10 9/9 0 100 0 not injected t1 0/1 11/11 0 100 9o 9 96 mode of injection; total number; neutralizing antibodies; number of tumours; of animals producing antibodies; of tumours; regression It is particularly noticeable that no animal treated in accoxlance with treatment c developed tumours as a consequence of RSV injection.

A series of further experiments with the aim of studying various parameters which can have an effect on the immune response, such as the preparation of the antigens, the route of immunization, the doses of antigen.., was carried out, restricting itself to the analysis of the production of neutralizing antibodies in the sera of the animals. These experiments were carried out, on the one hand, on adult animals, and, on the other hand, on 2-day old chicks.

1) On adult animals a) Influence of the way in which the antigens are prepared CEF/NEAs are irradiated and then harvested, either after trypsinization (treatment or by mechanical scraping of the unicellular layer and homogenization of the removed cells (treatment or, finally, by scraping and homogenization followed by ultrasound treatment 15 periods at a power of 40 W) of the cell suspension (treatment These cell preparations are deepfrozen and thawed before they are used.

Chickens aged 2 months (5 per treatment) are intravenously inoculated once with 107 cells per animal treated in accordance with treatments a, b and c. The ND 5 0 for each serum and each week was determined for 5 weeks after inoculation (Table III).

TABLE III Study of the effect of the antigen preparation in accordance with the treatments given above, on the effectiveness of immune response induction. The values represent the dilutions which permit neutralization of 50 of the NL virus.

TABLE III (3) 7d. 14d. 21d. 28d. (6) 1 Trypsin 0 0 0 1/4 1/4 2 0 0 0 1/4 1/8 3 0 0 0 1/8 '1/32 4 0 0 0 1/4 1/8 0 0 0 1/4 1/8 6 Scraping 0 1/128 1/512 1/256 1/128 7 0 1/128 1/512 1/512 1/128 8 0 1/128 1/1024 1/512 1/256 9 0 1/128 1/1024 1/2048 1/256 0 1/128 1/512 1/256 1/64 11 Scraping 0 1/128 1/2048 1/4096 1/1024 12 Ultrasound 0 1/128 1/2048 1/1024 1/128 13 0 1/128 1/1024 1/512 1/128 14 0 1/128 1/128 1/512 1/1024 0 1/128 1/2048 1/1024 1/512 serum; treatment of the antigen; seroneutralization.

16 It will be noticed that the highest ND 5 0 values which correspond to the most powerful immune responses are obtained with the antigen prepared in accordance with treatment Preparation induces a relatively powerful immune response, while the weakest immune response is found with the antigens which had been treated with trypsin.

2) Influence of the dose of antigen CEF/NE A which have been prepared in accordance with treatment above were used for intramuscular inoculation of chickens aged 3 months. These animals were divided into groups of 5 and inoculated with various doses of antigen, from 3 x 102 to 3 x 10 6 cells per animal (Table After 10 days, a second inoculation is carried out under the same conditions as the first. The sera are analyzed each week by means of seroneutralization, and the NDs 5 values between the various groups are compared (Table V).

TABLE IV Effect of the dose of antigen on the immune response The 2nd column gives the dose of antigen with which each animal is Lnoculated. The last 3 columns give the actual number of animale giving ND 5 s values which are higher or lower than the dilutions selected.

The results compiled in Table IV demonstrate that the immune response decreases with a decreasing dose of antigen inoculated. The inoculation of the weak dose of 3 x 10' cells per animal allows the determination of a weak humoral response which reaches ND 50 at the dilutions 1/8 to 1/32 35 days after the first inoulation.

17 Number of- Dose of 21d ND 50 animals antigen 28d 3x10 6 5> 1/8 5 1/32 5 >1/64 53x 4> 1/8 3>1/32 5>1/64 1: 1/4 2 1/32 3, o 4 3> 1/8 I 2>1/32 0>1/32 2> t/4 1/4 3x103 2= 1/8 5 1/4 not tested 3K<1/4 3) Influence of the route of inoculation with the antigen About 30 chickens aged 3 months and divided into 2 groups are inoculated with CEF/NEAs prepared in accordance with treatment At the point in time TO, the animals of the ist group are inoculated intravenously at a rate of 107 cells per animal, while the animals of tle 2nd group are inoculated intramuscularly. At T10, a booster is given under the same conditions. The sera of the animals are tested every week for their seroneutralizing capacity and the ND 50 for each serum determined. The numbers of animals of each of the 2 groups which allow an ND, 0 either higher than, or equal to, 1/32, or lowez than 1/32 to be obtained are compiled in Table VI. The results demonstrate that the effectiveness of inducing an immune response is better in the intravenous route compared with the intramuscular route. In fact, 8 animals out of 15 of group I (inoculated allow obtaining an

ND.

0 at a dilution higher than, or equal to 1/32 against only 2/14 in the second group It will be noted that whatever the route of immunization, all animals develop a humoral response with, however, variability as 18 regards, on the one hand, the latency period and, on the other hand,- the level of antibodies produced.

group number immunization ND 50 route higher than lower than 1/32 1/32 I 15 i.v. 8 7 II 14 i.m. 2 12 TABLE V Comparison between the effectiveness of inducing an immune response by the intravenous route and the intramuscular route.

4) Memorization of the humoral response At the conclusion of the analysis experiment on the above-described influence of the way in which the antigens are prepared, animals 1, 9 10; 14 and 15 (Table III) were kept. At T 9 gs after the 1st immunization, these animals were inoculated with 1D 7 cells of the CEF/NE

A

preparation of treatment The sera of the animals were harvested every other day for 2 weeks, and on days 21 and 28 after the last inoculation, and then tested by seroneutralization. The NDso values for each serum are compiled in Table VI and are used for plotting evolution curves for the humoral response as a function of time and immunization boosters.

These results demonstrate that following the 1st and 2nd inoculation with the antigen, the immune response develops progressively and slowly and reaches a maximum production of neutralizing antibodies 4 to 5 weeks after the 1st immunization. This response decreases and stabilizes at a mean value during the 2 months. After injecting the antigen at Tg 9 d, a very rapid production of the 77 neutralizing antibodies is observed. This production -19 reaches, or surpasses, the value of the maximum activity 2 to 3 times more quickly. These results demonstrate that this humnoral response is memorized.

.T ABLE VI :ND 50 value.

(6) 1 01 0 1/4 1/14 1/4 9 1/128 1/1024 1/2048, 1/256, 1/512 1/28, I/ I2 1/1,28 1/1024 1/128 14 11/128 1/128 1/512 1/1024 1/512 1/128 1/2048 1/1024 t/512 1/512 Table VI continuation: (9 (01 (1 2) (13) L/8 1/8 1/16 1/128 1/256 1/256 1/512 1/512 1/2048 1/4096 1/4 0 96 1/2048 1/256 1/256 1/1024 t/1024 1/1024 t/256 1/512 1/1024 1/8192 1/8192 1/8192 t/409 6 1/512 1/512 1/1024 1/2048 1/4096 1/4096 =anim~al; =14d; 21d; =28d; =3rd inoculation -95d; 97d; 99; =102; (10) =10611 (11) 116; (12) -116; (13) 123.

20 2) In 2-day old chicks 2-day old chicks are inoculated intraperitoneally with 10 viral particles of the NEA virus in a helper-free preparation (NEA-HF condition).

At points in time T3d and T6d, boosters under the same conditions as at the time of the first inoculation are given to all the animals.

At points in time T30d, T37d and T44d, blood samples are taken from all the animals, and the sera are harvested.

The results of the serum analysis by seroneutralization test demonstrate that all the sera exhibit a neutralizing activity against an avian retrovirus of subgroup A.

The ND,, value was determined for all the sera of sampling T44d.

In the case of the 20 chicks studied, 12 exhibited an ND 5 o value corresponding to a serum dilution lower than, or equal to, 1/1024, 4 to a dilution lower than, or equal to, 1/512, and the remaining four, lower than or equal to 1/8.

These results demonstrate that the NEA-HF virus is efficient in infecting chicks, since all the animals which had been inoculated at this stage by the NEA-HF preparation develop a humoral response which is represented by the neutralizing antibodies. The cells which are infected at the chick stage multiply and would determine an immune response producing neutralizing antibodies in the animal from 6-7 weeks, at the time of establishment of the immune system. However, this response is of varying intensity, depending on the individual.

It will seen that these results are different from those obtained in adult animals, since no humoral response or resistance to the development of tumours were observed in animals which had been treated at the adult stage in accordance with this method (for the method, see Table 2).

In the ease of ad'alt animals, the ineffectiveness 4 f infection with the NRA-HF virus could result from the ~9Q Y 21 rapid destruction of the infected cells by the immune system of the animal.

The results obtained from direct inoculation of 2-day old chicks with NEA-HF viruses might lead to a utilization of such a method including on 18-day old embryos with the aim of inducing resistance to infection by leucemogenic o sarcomatogenic avian retroviruses.

EXAMPLE 2: Immunizing composition against Newcastle disease 1. Construction of the vectors From laboratory-produced basic vectors pTXN3' and three constructions were carried out (vectors pNHnl, pNHn2 and pNHn3), and from he Hn gene which encodes for haemagglutinin neuraminidase of the Newcastle disease virus (NDV, strain Texas velogenic: Taylor et al Journal of virology 1990,64; 1441-1450) provided by Rh6ne-Mrieux. The fact that zhe HN gene is derived from strain Texas velogenic is not particularly important insofar as it is a model gene. One could just as well have used HN genes obtained from other strains. These constructions whose general structure and functioning are illustrated in Figure 1 differ from one another by the insertion pcsition of the HN gene in the basic retroviral vector, and by the processes which lead to the translation of the haemagglutinin neuraminidase protein, The details of the constructions will be given hereinafter.

Constructions of the plasmids SK+-HN-p and SK+-HN-P (see Figure 1) From plasmid pNDV71, which contains the Hn gene of the NDV (strain Texas), a 1.9 kb fragment containing this gene is isolated by double digestion with Scal/StuI.

This fragment is inserted into the filled-in SmaI and AccI sites of plasmid SK+. This gives plasmid SK+-HN-p, from which a 1.9 kb fragment which contains the Hn gene is isolated by digestion with Xhol, filling up, and SacI digestion. This gene is again sub-cloned in plasmid SK+ which had previously been digested with the enzymes EcoRV 22 and SacI, which leads to the formation of the plasmid SK+-NH-r.

Construction of the retroviral vector pHnN1: (see Figure 2) From plasmid SK+-HN-p, a 1.9 kb fragment containing the Hn gene is isolated by double digestion with XhoI/XbaI. This fragment is cloned into the Xhol and XbaI sites of the retoviral vector pTXN3' (which contains the neo gene in subgenomic position). This the vector pHnN1.

Construction of the retroviral vector pNHn2: (see Figure 3) From plasmid SK+-HN-p, a 1.9 kb fragment containing the Hn gene is isolated by double digestion with XhoI/Xbal, the ends of which are filled before it is cloned into the BglII site (filled-in) of the vector The resulting retroviral vector is named pNHn2.

Construction of the retroviral vector pNHn3: (see Figure 4) From plasmid SK+-HN-r, there is isolated by double XHoI/Xbal digestion, a 1.9 kb fragment which contains the Hn gene, of which the ends are filled, before it is cloned to the BglII site (filled-.n) of the vector pTXN5'. In the resulting retroviral vector pNHn3, insertion of the Hn gene from SK+-HN-r leads to the introduction of a stop codon on the sub-genomic RNA, which codon is in phase with the translation initiation codon of the gag gene. Moreover this insertion leads to the introduction of a spacer of about 70 nucleotides between this stop codon and the initiation codon of the Hn gene, which represents a distance favourable for optimum translation reinitiation.

The structures of vectors pNHnl, pNHn2 and pNHn3 are represented in Figure The vector chart pNDV71 is represented in Figure 6.

V

23 2. Production of v'ral stocks which are vehicles of the Hn gene Cell culture tests of these three vectors (immunocytochemistry or heamadsorption) revealed that these vectors allow expression of the HN protein. Vector pNHn3 allows authentic expression of the Hn protein (that is to say, without -eptide fusion in which is therefore fully functional. This is why this construction has been used for the ensuing work: production of viral stocks and vaccination assays.

Construction pNHn3 was transfected into the transcomplementing line Isolde. Polyclonal Isolde-NHn3 cultures were established which were capable of providing about 5 x 10 PFU/ml of supernatant. These t: ral stocks were studied after infection Cf cells of line QT6 or of CEF (chicken embryo cells). In all cases, the propagation of the vector NHn3 is exempt from that of a helper virus.

On neo+ cell colonies which are obtained after infection with the virus NHn3, heamadsorptions were carried out, and it was demonstrated that 80 of these clones can agglutinate erythrocytes.

3. Vaccination with the intermediary of cells presenting the, anti, n 3.1 Preparation of antigens associated with membranes The cells are prepared in the following manner: CEFs (or, in a second experiment, cells of the line LMH) are infected with a helper-free NHn3 viral stock, selected by G418, and, after neo+ colonies had appeared, amplified.

The cells are then rinsed (using PBS; phosphate-buffered saline), irradiated with UV (3 min, 253 nm) in order to destroy the genetic material, and harvested by scraping (or other methods in the case of the second experiment). These cells are then concentrated by centrifugation in such a way as to obtain about 10 cells per ml of PBS. These prenarations are then deep-frozen so that the membranes are lysed, and stocked in this manner.

24 3.2 Vaccination of adult animals Two experiments were carried out. The first one had the purpose of demonstrating what could be expected from such a procedure: we therefore started with a substantial quantity of recombinant cells (107 per aniial), injected with the most efficient adjuvant (Freund's Complete Adjuvant: FCA) and with a booster.

The second experiment had the purpose of refining the positive results of the first protocol end of improving them in the sense of a greater effectiveness of the method. This was carried out by testing various ways of preparing the antigen.

First experiment Groups of 10 chickens aged 3 weeks were immunized intraperitoneally with 107 cells per animal, using two injections at an interval of 15 days, and in the presence or absence of an adjuvant. The differences between the various groups relate to the addition of an adjuvant, namely its administration at the time of each injection, or only at the time of the booster, or, finally, in the absence of an adjuvant.

3 weeks after the booster, all animals are injected intraperitoneally with 10 53 viral particles of the Newcastlsedisease virus (NDV strain Texas) as virulent challenge.

The results obtained are given in Table 1. The effectiveness of the vaccination is given by the number of live animals 7 days after virulent challenge with the

NDV.

The treatment which imparts 100 of resistance to a virulent challenge corresponds to the one in which the recombinant cells were injected in the presence of FCA, simultaneously at the time of the initial injection and at the time of the booster. The two other treatments are only partially effective (10 to 20 effectiveness).

Group 5 consists of concrol animals which are not subjected to any immunizing injection.

Groups Nundbers First injectian Booster Number of% healthy subjects 1 10 10' CEFR 10 CEFR+FCA 1 2 10 10 7 CEFR 10 CEFR 2 3 9 10 CEFR+FCA 10' CEF 4 R+FCA 9 100 4 it6 10 CEFT 10 CEJ,'T+FCA 0 0 9 none none 00 quantity of cells injected. CEF R: CEF infe-ted with the vector NHn3, CEF T: non-infected CEFs, FCA injection in the presence of Freund's complete adjuvant.

TABLE 1: Anti-NDV vaccination: results of the first protocol 26 Second experiment: improvement of the presentation of the antigen In a second experiment, several immunization conditions were compared Table CEFs which were infected with viral stock NHn3 were prepared in a manner similar to the condition used at the time of the first experiment. This material was used for analysing: 1) the effect of a single injection in the presence of FCA (CEF FCA), and 2) the effect of another type of adjuvant (oily adjuvant: OA) by carrying out one single immunization injection (CEF OA).

The influence of a cell type other than CEFs was studied. To this end, cells of the line LMH (obtained from a chemically-induced chicken hepatoma Kawaguchi et al., 1990, Cancer Research, 47:4460-4464) were infected with the vector NHn3, selected by G418, and treated as in paragraph 3.1. This material was used for analysing: 3) the effect of these cells in the presence of OA adjuvant (LMH-OA) at the time of single or double booster injection, and 4) various preparation methods of these cells as immunization material: after trypsinization, after treatment with collagenase, after ultrasonic treatment of the clls which had been recovered by scraping (sonication), and finally after crushing them by ultraturax (crushing).

Checks for the effectiveness of the vaccination were carried out as in the first experiment: int.aperitoneal injection with the challenge virus NDV 3 weeks after the last vaccinating injection, and mortality checks (after 7 days) in the various immunized groups in relation to unvaccinated controls. Conditions 11, 12 and 13 represent controls: CEFs or LMH (12) not injected with the vector NHn3; and control animals which are not subjected to any injection (13).

j 27 Group Numbers I TO T15 poetdpoeto (fitst injection) 1 (booster) 1 recefPoeto

EF/

scraping

FCA

17CEF/ scraping

FAG

107 CEF/ scraping

FCA

107 CEF/ scraping FCa, 107

CEF/

scrap~ing

FCA

CEF/

scraping

OA

107 LHH/ tryps in

GA

107 LNM/ cotlagenase

OA

107 Lt4H/ sonicated

OA

107 LMH/ crushed

OA

107 LM11/ scraping

OA

107 LMH/ scraping

OA

11 10 iOCEF -0 0 control GA 12 10 10 7 LMH -0 0 control OA 13 10 0 0 FC~A :Freund's complete adjuvant, GA oily adjuvant.

TABLE 2 :Anti-NDV vaccination :second protocol 28 3.3 Conclusion Avian cells which express, on the surface, the HN protein of the Texas strain of the Newcastle disease virus were obtained by infection with the aid of a retroviral vector which serves as the vehicle, and which induces the expression of the Hn gene.

After the cells have been treated so as to destroy all nucleic acids and to break up the complete cells, they can, when chickens are injected, under certain conditions induce an immune response and a protection against a virulent challenge of NDV (10.3 viral particles, strain Texas).

The factors involved in a greater effectiveness of the protection seem to be the type of adjuvant used; 2) the cell type; 3) the alteration treatment of the membranes of the immunizing preparation.

The most efficient adjuvant is Freund's complete adjuvant, injected simultaneously at the time of a first immunization, and of a booster. Other types of adjuvants might be tested, even though, in principle, the oily adjuvant seems to be the one that is most adapted to avian vaccines.

The use of cells of liver cell line LMH which present the antigen HN, in comparison with the use of embryonic fibroblasts (CEP), seems to constitute an inmunizing material which is more favourable since, on the one hand, a single immunization with LMH-NHn3 entails a protection which is superior to that obtained with CEF-NHn3, and, on the other hand, as these cells are transformed and constitute a permanent line, is it then possible to indefinitely amplify the LMHNHn3, to deep-freeze them, and in this way to constitute a cell line of immunizing character.

The most effective treatment for obtaining protection is represented by the basic technique: the recombinant cells are harvested by scraping, subjected to UV action, and directly deep-frozen. Supplementary treatments, such as the action of proteolytic enzymes, ultra-sound treatment, or crushing them finely in an ultraturax, reduce the effectiveness of the preparations.

Claims

1. A process of preparing an immunizing compositio consisting of the combination of an immunogenic protein and cell membrane fragments, wherein said immunogenic protein is produced by the expression in suita,le host cells of a defective s vector for the integration and expression of a foreign gene coding for said protein in said host cells, said protein being produced by said host cells and presented in a form integrated into their membranes.

2. Process according to Claim 1, characterized in that said integration vector is a defective rctroviral vector which contains a selection gene and the gene for said 1o immunogenic protein, the vector being constructed in such a way as to optimize the production of said protein and so that the protein integrates itself into the membrane of the cells in which it is produced.

3. Process according to Claim 1 or 2, characterized in that said host cells are avian cells.

4. Process according to Claim 3, characterized in that said cells are CE or LMH chicken cells.

Process according to any one of Claims 1 to 4, characterized in that the composition also contains an adjuvant.

6, Process according to Claim 5, characterized in that the adjuvant is Freund's complete adjuvant.

7. Process for the preparation of a composition according to any one of Claims 1 to 6, characterized in that it comprises the following steps: a) the immunogenic protein is produced by expression, in appropriate host cells, of an integration and expression vector of a foreign gene encoding said protein in said 26 host cells, the vector being constructed in such a way as to optimize the production of said protein and so that the protein integrates iZself into the membrane of the cells Sin which it is produced, b) the host cells are subjected to a selection, in particular by the antibiotic which corresponds to S 0 $0 0 0 $0 30 the selection gene, and the cells which have thus been selected are multiplied, c) the resulting cells are treated in such a way as to inactivate the cell and viral nucleic acids which they contain, and d) the membrane constituents associated with the immunogenic protein are recovered and purified, if necessary.

8. Process according to Claim 7, characterized in that said integration vector is a defective retroviral vector which contains a selectiongene and the gene for said immunogenic protein.

9. Process for the preparation of an immunizing composition according to one of the preceding claims, characterized in that: a) a defective retroviral vector which contains a selection gene and the gene for the immunogenic protein is transfected on a culture of helper calls presented in the form of plasmid DNA. This results in the production of this same vector in the form of a defective virus which is capable of causing infection, b) with the helper-free viral preparation thus obtained, a culture of said host cells is infected, then c) the host cells are subjected to a selection, in particular by the antibiotic which corresponds to the selection gene, and the cells which have thus been selected are multiplied, d) the resulting cells are treated in such a way as to inactivate the cell and viral nucleic acids which they contain, and e) the membrane constituents associated with the immunogenic protein are recovered and purified, if necessary.

Process according to the preceding claim, charac- terized in that the viral vactors, the host cells and, -aee4 be- the helper-cells, are of avian origin. ^T\*a R9 A./ f^ R 1f 31

11. Process according to the preceding claims, characterized in that viral vector for the integration and the expression of the genome for the immunogenic protein in avian cells is composed, completely or partly, of the proviral genome of avian erythroblastosis or of a related virus in which the heterologous genes, namely the selection gene and the gene for the imnunogenic protein replace the genes v-erbA and v-erbB, and in that said genes are either under the control of an LTR promoter of the same virus, in which case the heterologous genes mime the genes which they replace, or under the control of a heterologous promoter, in which case an additional att sequence is positioned upstream of said heterologous promoter,

12. Process according to the preceding claim, charac- terized in that the viral vector is used for an inte- gration into chicken cells, such as CEF or LMH cells, and the heterologous genes are under the influence of the same avian LTR promoter and mime the genes which they replace.

13. Process according to the preceding claim, charac- terized in that, in the viral integration vector, the selection gene is located in the v-erbA position, and the gene corresponding to the immunogenic protein is located in the v-erbB position.

14. Process according to one of the preceding claims, characterized in that the heterologous genes are translated from the AUG of the gag gene, or from their own AUG, but always in the same reading frame as that of the gag gene.

Process according ti one of the preceding claims, characterized in that the selection gene is translated from the AUG of the gag gene, and the gene for the immunogenic protein is translated from the AUG of the gag gene or its own AUG.

16. Process according to one of the preceding claims, characterized in that a stop codon is introduced between the gag gene and the gene for the immunogenic protein or the AUG belonging to said gene for the immunogenic protein. i~ ""699= "1 r! 7

17. Process according to any one of the preceding claims, characterized in that the helper cells are QT6 quail cells obtained with the aid of a vector which is capable of transforming a normal QT6 cell into a helper cell containing all, or part, of the three genes gag-pol-env of the virus RAV-1 or RAV-2, placed under the transcriptional control of an LTR of the same virus on which various deletions have been carried out in order to suppress the capacity of the RNA produced by this vector for encapsidation.

18. A process for preparing an immunizing composition against the RSV virus according to anye ne of the preceding claims, characterized in that said protein is the env protein.

19. A process for the preparation of an immunizing composition against Newcastle disease according to any one of Claims 1 to 17, characterized in that said protein is the haemagglutinin neuraminidase (HN) of the Newcastle virus.

An immunizing composition, obtained by the process of any one of Claims 1 to is

21. A process of preparing an immunizing composition consisting of the combination of an immunogenic protein and cell membrane fragments which process is substantially as herein described with reference to Example 1 or 2,

22. A process for preparing an immunizing composition against the RSV virus which method is substantially as herein described with reference to Example 1.

23. A process for the preparation of an immunizing composition against Newcastle disease which process is substantially as herein described with reference to Example 2.

24, An immunizing composition whenever prepared by the process of any one of Claims 21 to 23.

25. A method of treating a viral disease in a host requiring said treatment, 25 comprising administering to said host an effective amount of an immunizing composition as defined in claim 20 or 24. 9 ."9 Dated 28 October, 1993 Institut National de la Recherche Agronomique Patent Attorneys for the Applicant/Nominated Person 30 SPRUSON FERGUSON *k 9 9 .9.9 4 ^.Es0 IG:\WPUSERLIBVVI00149:TCW 33 PATENT PROCESS FOR THE PREPARATION OF AN IMMUNIZING COMPOSITION INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE ABSTRACT The present invention relates to a process for the preparation of an immunizing composition which is composed of the association of an immunogenic protein and cell membrane fragments, said protein being produced by said cells and being presented in an integrated form with their membranes, characterized in that said immunogenic protein is produced by the expression, in suitable host cells, of a vector for integration and expression of a foreign gene which encodes for said protein in said host cells.