IMMUNIZING COMPOSITION AGAINST NEWCASTLE'S DISEASE AND METHOD OF PREPARATION.

Abstract

La présente invention a pour objet une composition immunisante contre la maladie de Newcastle constituée par l'association d'une protéine immunogène et de fragments membranaires de cellules, ladite protéine étant produite par lesdites cellules présentée sous forme intégrée à leur membrane caractérisée en ce que la dite protéine provient du virus de Newcastle, il s'agit notamment de l'hémaglutinine-neuraminidase.. La présente invention a également pour objet un procédé de préparation d'une telle composition à l'aide de vecteurs rétroviraux.

Description

The present invention relates to an immunizing composition against the Newcastle disease and a preparation method using vectors for integrating and expressing a foreign gene in host cells. This certificate of addition constitutes a development of main patent application NO. 89 12 764 filed on September 29, 1989, to which reference should be made for a better understanding of the present invention. More specifically, the present invention relates to an immunizing composition against Newcastle disease consisting of an immunogenic protein from the Newcastle virus and cell membrane fragments, said protein being produced by said cells and subsequently integrated into their membranes. According to another highly advantageous feature of the present invention, the immunogenic protein is produced by the expression in appropriate host cells of an integration and ex- vector pressure of a gene encoding said protein in said host cells. More specifically, the said integration and ex- vector pressure will be a defective retroviral vector comprising a selection gene and the gene for said immunogenic protein, this vector being constructed in such a way as to optimize the production of the protein and so that it integrates to the cell membrane in which it is produced. In one embodiment of the invention, the protein is the Newcastle virus hemagglutinin-neuraminidase (HN), in particular a protein produced from a gene originating from the Texas strain Newcastle disease virus velocity. Appropriately, the protein is produced from an avian retroviral vector and in avian cells, in particular chicken CEF or LMH cells. Compositions according to the invention They therefore contain membrane fragments of said cells. Furthermore, the compositions according to the invention include advantageously an adjuvant, in particular Freund's complete adjuvant. The present invention also relates to a method for preparing a composition, characterized in that a) the immunogenic protein is produced by the expression in appropriate host cells of an integration vector and the expression of a foreign gene encoding said protein in said host cells, the vector being constructed so as to optimize the production of said protein and so that it integrates into the membrane of the cells in which it is produced, b) host cells are subject to selection, notably by antibiotics a) the tick corresponding to the selection gene is identified, and the cells thus selected are multiplied; c) the resulting cells are treated to inactivate the cellular and viral nucleic acids they contain; d) the associated membrane components are recovered and purified as needed. to the immunogenic protein. In a particular embodiment of a method for preparing an immunizing composition according to the invention, the method comprises the following steps: production of a defective retroviral vector presented in the form of plasmid DNA comprising a selection gene and the gene for an immunogenic protein, the vector being constructed so as to optimize the production of the immunizing protein, the structure of this protein and of the integration vector being constructed such that the protein integrates into the membrane of the cell in which this protein is produced; transfection of this vector onto a culture of helper cells from which results the production of a viral preparation of this same vector in the form of a defective virus endowed with infectious capacity; the viral preparation obtained is used to infect a culture of ordinary cells so that no transmissible virus can be formed;the cells are then subjected to selection by the antibiotic corresponding to the selection gene used according to the procedure known to those skilled in the art; the cells thus selected are multiplied according to a classic procedure; the cells obtained are treated in such a way as to inactivate the cellular and viral nucleic acids they contain, in particular by UV irradiation; then freezing and thawing, and we recover the membrane constituents. compounds associating the immunogenic protein, constituents which are purified at need. This preparation method has proven particularly effective and has never before been used in the preparation of a vaccine. In one particular embodiment, the following are used: avian vectors that are cultured on avian cells. An immunogenic protein produced on avian cells and presented on membrane fragments of said avian cells exhibits considerably enhanced immunogenic capabilities, even on mammals in relation to said purified protein. The use of an avian retroviral vector to prepare such a protein allows for perfect control of the integration process of the immunogenic protein gene and the production of said protein in avian cells. This allows for the optimization of all elements such as transcriptional activity, protein production, and the presentation of this protein on receptors capable of amplifying its immunogenic activity. All these results are more difficult to obtain by a routine transfection. Of course, the use of an avian vector requires the culture of avian cells. However, the same result can be obtained with vectors and cells of another origin, for example, a mammalian vector cultured on mammalian host cells, such as a murine vector. Viral vectors for the integration and expression of a heterologous gene in avian cells have been described in European and international patent applications EP 178 996 and PCT / FR 88 00487 (WO I Lt 89 / 03877). Refer to these applications for a better understanding of the... present patent application and more specifically for the description detailed description of useful avian viral vectors according to the present invention in particular the vectors p TXN 3 ' and p TXN 5 '. In a particular embodiment of the invention, a vector consisting of all or part of the proviral genome of avian erythroblastosis or of a related virus will be used as a vector for integrating and expressing the immunogenic protein gene in avian cells, in which the immunogenic protein gene and the selection gene replace the v-erb A and v-erb B genes, and in which said genes are either under the control of an LTR promoter of the same virus, in which case said genes mimic the genes they replace, or under the control of a heterologous promoter, in which case an att sequence additional is positioned upstream of said heterologous promoter gene. The avian viruses most particularly relevant to the present invention are P'AEV and related viruses of the ALSV type, as well as than non-defective viruses of the RAV type. In a useful embodiment, particularly for integration- In chicken cells, such as CEP chicken embryo fibroblasts or LMH liver cells, the immunogenic protein gene is dependent on avian LTR and their the translation mechanism mimics that of the genes they replace. Appropriately, the vector carries the selection gene at position v-erb A, and the immunogenic protein gene at position v-erb B. Advantageously, according to another characteristic of these vectors, the selection gene and the immunogenic protein gene are translated from the AUG of the gag gene or from their own AUG, but still within the same reading framework as that of the gag gene. Thus, where applicable, the selection gene is translated from the AUG of the gag gene and the immunogenic protein gene is translated from 1 'AUG of the gag gene or its own AUG. Advantageously, a stop codon is introduced between the gag gene and the immunogenic protein gene or the AUG specific to said gene of the immunogenic protein. Preferably, the stop codon is located approximately 70 nucleotides from the AUG codon of said immunogenic protein gene, in particular 65. In another embodiment of the invention, the integration and expression vector is such that the selection gene is in the v-erb A position under the control of an avian LTR 5' promoter and the immunogenic protein gene is in the v-erb B position under the control of a heterologous promoter, the vector then comprising an att sequence additional positioned upstream of the heterologous promoter. In vectors that have an additional att sequence, a portion of the U5 sequence of the 5' LTR can be deleted such that after a retroviral cycle only the att sequence remains functional. additional which ensures integration. Preferably, a 23 bp deletion is performed in the region 3' terminal of the U 5 sequence of the LTR 5'. Advantageously, the vector is characterized in that the immunogenic protein gene is in the v-erb B position flanked by the heterologous promoter and the heterologous polyadenylation sequence, and in that the entire promoter-immunogenic protein gene-polyadenylation sequence is in the same or opposite orientation to the retroviral transcriptional meaning. One example that can be cited as a heterologous promoter is promoter of the simian virus SV 40. The implementation of the process according to the invention also requires the use of permanent helper cell lines capable of producing helper-free viral preparations. These helper cell lines provide the defective genome with the gag-pol-env proteins necessary for it to form virions and thus enable to use defective viral vectors without the addition of helper viruses. According to the present invention, a vector capable of transforming a normal avian cell into a "helper" cell may be used, in particular, a vector comprising all or part of the three gag-pol-env genes of the RAV-1 or RAV-2 virus placed under the transcriptional control of an LTR of the same virus to which various deletions have been introduced in order to suppress the ability to encapsidate P'RNA produced by this vector. In one embodiment, cells are used departure to prepare the helper cells, QT 6 quail cells. In one embodiment, particularly when the integration and expression vector includes the env gene as the immunizing protein gene, it was possible to remove the following from the vector intended to transform the normal cells into "helper" cells the gene env. In other embodiments, the gag and poi genes can be deleted and replaced by a selection gene, the env gene. subsisting being preceded by its splicing acceptor site. As mentioned previously, other characteristics of avian vectors useful in the process will be described according to the invention in international patent application WO 89 / 03877. Other features and advantages of the present invention will appear in light of the detailed description of the modes of The following achievements. Figure 1 represents the construction of the SK Hn- vector Figures 2 to 4 represent the constructions of the vectors. p Hn N, p Hn N 2 and p Hn N 3. Figure 5 represents the vector structure of Figure 2 to 4 carrying the Hn gene of NDV. Figure 6 represents the map of the p NDV-71 plasmid: ATG: Translation initiation codon; TGA: Translation terminator codon; Amp R, Tcl resistance genes, carried by the p BR 322 sequence, to the antibiotics ampicillin, Tetracycline; ori: origin of plasmid replication F, L: residue of the F and L genes of NDV. Hn: Hn gene poly AF, poly A Hn: polyadenylation sequences messengers of the F and Hn genes. The large boxes represent the Rs T cells, the Hn gene, and the selection gene. The small boxes represent retroviral gene residues. The thin lines represent RNAs transcribed from vectors, while the thick lines represent synthesized proteins. The white or black triangles represent translation start or stop codons. ds and as are the donor and splicing acceptors. 1. Vector Construction Three constructions were carried out (vectors pNHn1, pNHn2, and pNHn3) from the base vectors pTXN3' and pTXN5' produced in the laboratory, and from the Hn gene encoding the hemagglutinin-neuraminidase of Newcastle disease virus (NDV, Texas velocipede strain: Taylor et al., Journal of Virology 1990;64:1441-1450) provided by Rhône-Mérieux. The fact that the HN gene comes from the Texas velocipede strain is not particularly important since it is a model gene. HN genes from other strains could just as easily have been used. These constructions, whose general structure and function are illustrated in Figure 1, differ from each other by the insertion position of the Hn gene in the base retroviral vector. than by the processes leading to the translation of the hemag- protein glutinin-neuraminidase Construction details are shown below. Construction of SK+-HN-P and SK+-HN-f plasmids: (see Figure 1) A 1.9 kb fragment containing the Hn gene from the NDV p plasmid (Texas strain) is isolated by double Scal / Stul digestion. This fragment is inserted into the filled Smal and Accl sites of the SK+ plasmid. This results in the SK+-HN-P plasmid, from which a 1.9 kb fragment containing the Hn gene is isolated by Xhol digestion, filling, and Sacl digestion. This fragment is then subcloned back into the SK+ plasmid, which has been previously digested with the Eco RV and Sacl enzymes. leads to the formation of the SK+-HN-r plasmid. Construction of the p Hn Nl retroviral vector: (see Figure 2) A 1.9 kb fragment containing the Hn gene is isolated by Xhol / Xbal double digestion from the SK+-HN-P plasmid. This fragment is cloned into the Xhol and Xbal sites of the p TXN 3' retroviral vector (containing the (neo gene in subgenomic position) This results in the p Hn Nl vector. Construction of the pN Hn 2 retroviral vector: (see Figure 3) A 1.9 kb fragment containing the Hn gene with filled ends is isolated by double Xhol / Xbal digestion from the SK+-HN-P plasmid before cloning it into the filled Bglll site of the The resulting retroviral vector is called p TXN 5'. Construction of the pNH3 retroviral vector: (see Figure 4) A 1.9 kb fragment containing the Hn gene is isolated by double Xhol / Xbal digestion from the SK+-HN-P plasmid. The ends of this fragment are filled before cloning it into the filled Bglll site of the pTXN5' vector. In the resulting pNH3 retroviral vector, the insertion of the Hn gene from SK+-HN introduces a stop codon, in phase with the translation start codon of the gag gene, onto the subgenomic RNA. Furthermore, this insertion introduces a spacer of approximately 70 nucleotides between this stop codon and the Hn gene start codon, representing a distance favorable for optimal reinitiation of translation. The structures of the pNH3, pNH2, and pNH3 vectors are shown. figure 5. The map of the vector p NDV 71 is shown in Figure 6. 2. Production of viral stocks carrying the Hn gene. Cell culture tests of these three vectors (by immunocytochemistry or hemadsorption) showed that these vectors allow expression of the HN protein. The pNH3 vector allows authentic expression of the Hn protein (i.e., without peptide fusion at the 5' end), which is therefore fully functional. This is why this construct was used for subsequent work: production of viral stocks, and vaccination trials. The p N Hn 3 construct was transfected in the transcom- line Using Isolde I 1, polyclonal Isolde-N Hn 3 cultures were established, capable of producing approximately 5 x 10 RFU / ml of supernatant. These viral stocks were studied after infection of QT6 cell lines or CEF (chicken embryo cells). In all cases, the propagation of the N Hn 3 vector was free from that of a helper virus. Hemadsorptions were performed on neo+ cell colonies obtained after infection with the N Hn 3 virus, and it was shown that 80% of these clones can to agglutinate erythrocytes. 3. Vaccination via antigen-presenting cells 3.1 Preparation of membrane-associated antigens The cells are prepared as follows: Fs ECs (or MHL cells in a second experiment) are infected with a helperfree N Hn 3 viral stock, selected with G 418, and amplified after the appearance of neo+ colonies. The cells are then rinsed (with PBS - Phosphate Buffered Saline-), irradiated with UV light (3 min, 253 nm) 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 to obtain approximately 10 cells per ml of PBS. These preparations are then frozen to lyse the membranes. and thus stored. 3.2 Vaccination of Adult Animals Two experiments were conducted. The first aimed to demonstrate the potential results of such a procedure: we therefore started with a large quantity of recombinant cells (10⁷ per animal), injected with the most effective adjuvant (Freund Complete Adjuvant: CFA), and with a booster dose. The second experiment aimed to refine the positive results of the first protocol and improve them to achieve greater methodological efficiency. This was done by testing various modes of antigen preparations. 1. First experiment. Groups of 10 three-week-old chickens were immunized intraperitoneally with 10⁷ cells per animal, in two injections 15 days apart, with or without adjuvant. The differences between the various groups concerned the addition of adjuvant, namely its administration with each injection, or only with the booster, or finally, in the absence of an adjuvant. Three weeks after the booster, all animals are injected intraperitoneally with 10⁵ ³ viral particles of the virus of the disease Newcastle (NDV Texas strain) as a virulent test. The results obtained are presented in Table 1. Vaccination efficacy is represented by the number of animals alive 7 days later. after the virulent ordeal by the NDV. The treatment providing 100% resistance to a virulent challenge corresponds to the one in which recombinant cells were injected in the presence of ACF, simultaneously during the initial injection and the booster. The other two treatments are only partially effective (10 to 20% efficacy). Group 5 consists of animals controls not receiving any immunizing injection. Primary injection sample sizes recall numbers (a) (a) healthy subjects 1 10 107 CEFR 107 CEFR+ACF I 10 2 10 107 CEFR 107 CEFR 2 20 3 9 107 CEFR+ACF 107 CEFR+ACF 9 100 4 10 107 CEFT 107 CEFT+ACF OO 9 nothing nothing OO (a): quantity of injected cells CEF R: CEF infected by the vector N Hn 3, CEF T: CEF uninfected, ACF: injection in the presence of Freund's complete adjuvant. TABLE 1: Anti-NDV vaccination: results of the first protocol Co cq cl rl OO ol (N Second experiment: improvement of antigen presentation In a second experiment, several immunization conditions were compared (see Table 2). CE Fs infected with the viral stock N Hn 3 were prepared similarly to the condition used in the first experiment. This material was used to analyze: 1) in the presence of ACF, the effect of a single injection (CEF ACF) 2) the effect of another type of adjuvant (oily adjuvant: AH) in practicing a single immunization injection (CEF AH). The influence of a cell type other than Fs CE was studied. For this purpose, LMH cell lines (derived from a chemically induced chicken hepatoma, Kawaguchi et al., 1990, Cancer Research, 47:4460-4464) were infected with the NH3 vector, selected with G418, and treated as described in paragraph 3.1. This material was used to analyze: 3) the effect of these cells in the presence of AH adjuvant (LMH-AH) during single or double injection as a booster. 4) various methods of preparing these cells as immunization material: after action of trypsin, collagenase, after action of ultrasound ("sonicated") on cells recovered by scraping, finally after grinding ("ground") by ultraturax. Vaccination efficacy tests were performed as in the first experiment: injection of the NDV test virus, 3 weeks after the last intraperitoneal vaccination, and monitoring of mortality (after 7 days) between the different groups immunized compared to unvaccinated controls. Conditions 11, 12, and 13 constitute the controls: CE Fs (11), or LMH (12) not infected by the N Hn 3 vector; and control animals not undergoing no injection ( 13). Effective Groups TO (primary injection) T 15 (reminder) protegesprotection 107 CEF / scraping ACF 107 CEF / scraping ACF CEF / scraping ACF CEF / scraping ACF CEF / scraping ACF CEF / scraping AH LMH / trypsin AH LMH / collagenase AH LMH / sonicated AH LMH / crushed AH LMH / scraping AH LMH / scraping AH FO 11 10 11 l 10 10 CEF witnesses _ OO 7 AH 12 10 10 LMH witnesses OO AH 13 10 OO ACF: Freund's complete adjuvant, AH: oily adjuvant. TABLE 2: Anti-NDV vaccination: second IO protocol 1 1 i I' 3.3 Conclusion Avian cells expressing on their surface the HN protein of the Texas strain of Newcastle disease virus were obtained by infection using a retroviral vector carrying, and inducing the expression of the Hn gene. After treatment to destroy all nucleic acids and break down all cells, these, when injected into chickens, can, under certain conditions, induce an immune response and protection against a virulent challenge of NDV (105-3 viral particles). of the Texas strain). Factors influencing greater protective efficacy appear to be: 1) the type of adjuvant used; 2) the cell type; 3) the treatment of membrane alteration of the immunizing preparation. The most effective adjuvant is Freund's complete adjuvant, injected simultaneously during primary immunization and a booster dose. Other types of adjuvant could be tested, although in principle the adjuvant Oily seems to be the most suitable for avian vaccines. The use of LMH liver cell lines expressing the HN antigen, compared to the use of embryonic fibroblasts (EFs), appears to be a more favorable immunizing material since, on the one hand, a single immunization with LMH-N Hn 3 results in greater protection than that obtained with EFS-N Hn 3, and on the other hand, since these cells are transformed and constitute a perennial line, it is then possible to amplify LMH-N Hn 3 indefinitely, freeze them, and thus create an immunizing cell line. The most effective method for obtaining protection is represented by the basic technique: recombinant cells are harvested by scraping, exposed to UV radiation, and immediately frozen. Additional treatments such as the action of proteolytic enzymes, sonication by ultrasound, or fine milling with an ultrasonic cleaner reduce the effectiveness of the preparations.

Claims

DEMANDS 1. Immunizing composition against Newcastle disease according to one of claims 1 to 3 of the main patent characterized in this that it is constituted by the association of an immunogenic protein from the Newcastle disease virus and cell membrane fragments, said protein being produced by said cells and presented in a form integrated into their membranes; 2. Composition according to claim 1, characterized in that said protein is the hemagglutinin-neuraminidase (HN) of the Newcastle virus; 3. Composition according to claim 1 or 2, characterized in that the immunogenic protein is produced by the expression in suitable host cells of an integration and expression vector of a foreign gene encoding said protein from the Texas strain Newcastle disease virus velocity in said host cells. 4 Composition according to claim 2, characterized in this that said cells are avian cells. Composition according to claim 3 characterized in that These cells are CEF or LMH chicken cells. 6 Composition according to any one of claims 1 to 4 character- sée in that it also includes an adjuvant. 7 Composition according to claim 5 characterized in that the adjuvant is Freund's complete adjuvant.

8. Method for preparing a composition according to one of the claims 1 to 3, characterized in that a) The immunogenic protein is produced by the expression in appropriate host cells of an integration vector and the expression of a foreign gene encoding said protein in said host cells, the vector being constructed so as to optimize the production of said protein and so that it integrates into the membrane of the cells in which it is produced, b) host cells are subject to selection, notably by antibiotics a) the tick corresponding to the selection gene is identified, and the cells thus selected are multiplied; c) the resulting cells are treated to inactivate the cellular and viral nucleic acids they contain; d) the associated membrane components are recovered and purified as needed. to the immunogenic protein.

9. A method according to claim 4, characterized in that said integration vector is a defective retroviral vector comprising a gene selection and the gene for said immunogenic protein. Method for preparing an immunizing composition according to one of the preceding claims, characterized in that: a) A defective retroviral vector containing a selection gene and the immunogenic protein gene is transfected into a culture of helper cells presented as plasmid DNA. This results in the production of the same vector as a defective virus with infectious capabilities. b) A culture of said host cells is infected with the helper-free viral preparation thus obtained, then c) Host cells are subject to selection, notably by antibiotics. a tick corresponding to the selection gene, and the cells thus selected are multiplied, d) the resulting cells are treated to inactivate the cellular and viral nucleic acids they contain, e) the associated membrane constituents are recovered and purified as needed to the immunogenic protein. The method according to the preceding claim, characterized in that the viral vectors, host cells, and helper cells where applicable, are of avian origin.

12. Method according to the preceding claims, characterized in that the viral vector for integration and expression of the immunogenic protein gene in avian cells consists of all or part of the proviral genome of avian erythroblastosis or a related virus in which the heterologous genes, namely the selection gene and the immunogenic protein gene, replace the v-erb A and v-erb B genes, and in that said genes are either under the control of an LTR promoter of the same virus, in which case the heterologous genes mimic the genes they replace, or under the control of a heterologous promoter, in which case an additional att sequence is positioned upstream of said heterologous promoter.

13. A method according to the preceding claim, characterized in that the viral vector is used for integration into chicken cells such as CEF or LMH cells and the heterologous genes are under the control of the same avian LTR promoter and They mimic the genes they replace. 14 Method according to the preceding claim, characterized in that in the viral integration vector, the selection gene is located at position v-erb A and the gene corresponding to the immunogenic protein is located at position v-erb B. A method according to any one of the preceding claims, characterized in that heterologous genes are translated from the AUG of the gag gene or from their own AUG but always in the same reading framework than that of the gag gene.

16. Method according to any one of the preceding claims, characterized in that the selection gene is translated from the AUG of the gag gene and the immunogenic protein gene is translated from the AUG of the gag gene or its own AUG.

17. A method according to any one of the preceding claims, characterized in that a stop codon is introduced between the gag gene and the immunogenic protein gene or the AUG proper to said immunogenic protein gene. u ' 2663228 18 Method according to claim 8, characterized in that the The retroviral vector is the pNH3 vector shown in Figures 4 and 5.

19. Method according to any one of the preceding claims, characterized in that the helper cells are QT6 quail cells obtained using a vector capable of transforming a normal QT6 cell into a helper cell containing all or part of the three genes gag-pol-env of the RAV 61 or RAV-2 virus, placed under transcription- control nel of an LTR of the same virus to which various deletions have been made in order to suppress the ability to encapsidate the RNA produced by this vector.