WO1993019092A1

WO1993019092A1 - Defective recombinant adenoviruses expressing characteristic epstein-barr virus proteins

Info

Publication number: WO1993019092A1
Application number: PCT/FR1992/000256
Authority: WO
Inventors: Michel Perricaudet; Thierry Ragot; Susan Finerty; Andrew J. Morgan
Original assignee: Centre National De La Recherche Scientifique; Cancer Research Campaign In London
Priority date: 1992-03-19
Filing date: 1992-03-19
Publication date: 1993-09-30
Also published as: AU1648092A

Abstract

A recombinant vector characterized in that it includes a genomic sequence of a defective adenovirus, i.e. one that lacks the sequences needed for it to replicate, as well as an insert including one or more DNA sequences coding for any characteristic protein of the Epstein-Barr Virus (EBV) which can induce the formation in a patient of EBV-protective neutralizing antibodies and, if required, protective cells, particularly cytotoxic and helper lymphocytes, said insert being under the control of a promoter present in or pre-inserted into the adenovirus genome. Said vector may be used to produce vaccines for diseases caused by EBV in humans or animals.

Description

DEFECTIVE RECOMBINANT ADENOVIRUSES EXPRESSING PROTEINS

CHARACTERISTICS OF EPSTEIN-BARR VIRUS

The subject of the invention is recombinant adenoviruses defective for replication expressing glycoproteins characteristic of the Epstein-Barr virus, in particular major envelope glycoproteins of this virus, as well as the use of these recombinant adenoviruses in vaccine compositions directed against pathologies resulting from an individual's infection with the Epstein-Barr virus (EBV).

The Epstein-Barr virus is present in a latent state in a fraction of the B lymphocytes of the vast majority of adults. This herpes virus is responsible for infectious mononucleosis and is etiologically associated with endemic Burlcitt lymphoma and undifferentiated carcinoma of the nasopharynx (NPC) (Epstein and Achong, 1979, 1986). It is also involved in lymphomas appearing in immunocompromised patients, such as individuals who have undergone an organ transplant or patients with AIDS (Thomas and Crawford, 1989). More recently this virus has been associated with certain types of Hodgkin's lymphoma (Mueller et al., 1989; Pallesen et al., 1991).

The prevention or control of these diseases by vaccination has been the subject of much research (Epstein, 1976), especially in the case of NPC which reaches up to 2% of the male population in southern China and other high incidence regions such as Southeast Asia or medium incidence such as North Africa.

EBV therefore represents a major health problem worldwide. Unfortunately, it is not possible to use EBV as a live vaccine because it replicates very weakly in vitro and transforms human B lymphocytes in vivo.

The major envelope glycoproteins of EBV, namely the gp340 and gp220 proteins, both originate from the same viral gene by a splicing phenomenon without changing the reading frame (Beisel et al., 1985), and are expressed on the outer surface of virions and on the membrane of infected cells. These proteins bind to the CR2 cellular receptor (CD21) of the complement C3d component (Fingeroth et al., 1984; Tanner et al., 1988) and are capable of inducing the formation of EBV neutralizing antibodies in humans. (Thorley-Lawson and Poodry, 1982). Monoclonal antibodies against gp340 / 220 also neutralize the virus in vitro (Hoffman et al., 1980). These glycoproteins induce a cellular response (T cells) (Uleato et al., 1988; Bejarano et al., 1990) as well as an antibody-dependent cell cytotoxicity (Qualtière et al., 1982) and also induces the formation of antibodies. neutralizers.

Cottontop tamarin monkeys have been successfully protected against EBV-induced lymphoma by a gp340-based vaccine purified from B95 / 8 cells (Epstein et al., 1985; Morgan et al., 1988a and 1989). However, the quantity of gp340 directly obtained from EBV is too insufficient to allow consideration of the use of proteins purified from EBV as a vaccine. On the other hand, the preparations obtained can be contaminated with EBV DNA, which is not acceptable in human vaccination.

The use of vaccines based on recombinant EBV proteins obtained by transformation of bacteria or yeasts by nucleotide sequences coding for these proteins has encountered the problem of the absence of glycosylation or glycosylations different from the polypeptides obtained from those normally seen in mammalian cells. These proteins are therefore weakly immunogenic or induce inappropriate immune responses in rabbits (Emini et al., 1988).

Recombinant vectors have been made using recombinant viral vectors expressing the gene encoding gp340 / gp220, such as vaccinia virus (Mackett and Arrand, 1985) and varicella-zoster virus (Lowe et al., 1987). Protection against EBV-induced lymphoma was achieved using a vaccinia virus laboratory strain (WK) as a vector, but was not achieved using a more attenuating vaccine strain ( Wyeth) (Morgan et al., 1988b). However, only one attenuated strain of the vaccinia virus can be used in the context of a vaccination against EBV using recombinant vaccinia, which consequently limits the effectiveness of this type of vaccine.

One of the aims of the present invention is to provide vaccinating compositions against the pathologies caused by the infection of an individual by EBV, which have both the advantage:

- to be able to be produced in sufficient quantity at an advantageous cost price,

- to effectively protect individuals against EBV by conferring cellular immunity ("helper" T cells and cytotoxic) and by production of protective antibodies against EBV,

- not to risk contaminating the environment of individuals treated with these compositions and therefore to be able to be used safely.

The subject of the present invention is recombinant nucleic acids characterized in that they comprise, on the one hand, a genomic sequence of a defective adenovirus, that is to say devoid of the sequences necessary for its replication, and, on the other hand on the other hand, an insert comprising one or more DNA sequences coding for any protein characteristic of EBV which is capable of inducing in an individual the formation of protective neutralizing antibodies against EBV and, where appropriate of protective cells, in particular cytotoxic T cells and T helper cells, this insert being placed under the control of a promoter present or previously inserted into the genome of the adenovirus.

Advantageously, the genomic sequence of the abovementioned defective adenovirus nevertheless includes those of the sequences which in this genome are the carrier of the genetic information necessary for the corresponding adenovirus to penetrate cells which can be infected with it, and, the if necessary, all of the essential sequences necessary for the packaging of this adenovirus.

The invention relates more particularly to the use of these nucleic acids as vectors for the transformation of human or animal cells.

As such, the promoter, under whose control the above-mentioned insert is placed, is advantageously likely to be recognized by the polymerases (and more particularly the polymerases II) of human or animal cells infectable with this virus (representing a large number of different cell types), this promoter being in particular the late major major promoter (MLP) of human adenovirus type 2 (Levrero et al., 1991) or any other known ubiquitous promoter.

Adenoviruses, in particular adenoviruses of type 2 or 5 (Ad2 or Ad5), represent particularly preferred vectors within the framework of the present invention, for the following reasons: in general, adenoviruses are relatively stable, can be cultured easily and rapidly (30 h viral cycle), give particularly high titers (up to 104 to 105 plaque forming units or pfu per infected cell). Types 2 or 5 are more particularly preferred since these serotypes are not oncogenic in rodents such as, for example, serotype 7, the complete sequence of their viral genome has been established (Chrobozek et al., 1992), their molecular biology has been intensively studied, finally, many mutants, in particular deletion mutants have been obtained, making it possible to insert large DNA fragments (Berkner, 1988).

Advantageously, the DNA sequence (s) mentioned above, coding for any protein characteristic of EBV, is or are included in a defective adenovirus genome, this genome being devoid of the essential nucleotide sequences necessary the replication of this virus in permissive cells, and more particularly sequences coding for the E1 region, and, where appropriate for the E3 region. This E1 region comprises the immediately early gene E1A which activates the other early transcription units of the virus and is therefore necessary for its replication, and the E1B gene which is responsible for the complete transformation of cells in culture in the presence of E1A. The defective genome adenovirus nevertheless preferably comprises all of the essential sequences necessary for the constitution of a viral stock on cells (293 cells) complementing the E1 region (E1A + E1B) of the virus.

According to a particularly advantageous embodiment of the invention, the above-mentioned foreign DNA sequences are inserted in place of the E1 region of the Ad5 genome, a region allowing the insertion of several genes coding for immunogenic proteins .

Preferably, the genome of the recombinant vectors according to the invention comprises a DNA sequence coding for a major envelope glycoprotein of EBV, and more particularly for the glycoprotein gp340 or gp220 of EBV, or for any fragment of these. proteins which is capable of inducing the formation of protective antibodies against EBV in an individual, and, where appropriate, of protective cells of the immune system, in particular cytotoxic T cells and T helper.

Other particularly advantageous recombinant vectors in the context of the present invention are those whose genome comprises a DNA sequence coding for an EBV membrane antigen involved in the mechanism of fusion of the EBV viral envelope with the membrane of B lymphocytes and epithelial cells, and more particularly the glycoprotein gp85 (Haddad et al., 1989), or for any fragment of this protein capable of inducing the formation of protective antibodies or protective cells as defined above.

Preferred vectors according to the invention are those comprising both an insertion nucleic acid encoding a major envelope glycoprotein of EBV, and more particularly for the glycoprotein EBV gp340 or gp220, or for any fragment of these proteins as defined above, and an insertion nucleic acid encoding an EBV membrane antigen involved in the fusion mechanism of the viral envelope EBV with the cell membrane of the body's cells, and more particularly the glycoprotein gp85 or for any fragment of this protein as defined above.

Advantageously, the genome of the vectors according to the invention also comprises a DNA sequence coding for a protein stimulating the immune system, and more particularly the cellular response (cytokines).

As such, the present invention relates to vectors as described above and whose genome comprises a DNA sequence coding for interleukin-2, interleukin-3, interleukin-4, interleukin -5, interleukin-6, interleukin-7, or any fragments thereof that may stimulate the immune system.

The invention also relates to pharmaceutical compositions comprising one or a combination of several recombinant vectors as described above, in combination with a pharmaceutically acceptable vehicle (adjuvants, immunostimulatory complexes).

The invention more particularly relates to vaccines directed against pathologies caused by EBV in humans or animals, these vaccinating compositions comprising one or more recombinant vectors as described above, in combination with a pharmaceutically carrier acceptable.

As such, the present invention relates to methods of prevention, mitigation or treatment of pathologies caused by EBV in humans or animals, these methods comprising the administration in humans or animals likely to be infected with EBV or at risk of being infected, of a vaccine or of a pharmaceutical composition according to the invention.

The use of non-replicable adenoviruses in the context of the present invention offers the advantage on the one hand of avoiding contamination of the environment of the individuals treated with the aid of these vectors, and, on the other hand, to avoid obtaining in the treated individuals too strong immunization against the proteins characteristic of the adenovirus, and more particularly against the proteins of the capsid (hexons and fiber), thus authorizing the carrying out of several immunizations in complete safety.

The quantity of vectors administered into the organism is advantageously chosen so as to provoke an appropriate immune response directed essentially against the vaccinating antigens inserted into the viral vector in the organism into which they are injected.

Advantageously, the routes of administration chosen in the context of the present invention are the intramuscular, intranasal or the oral route in the form of gastro-protected capsules capable of delivering the viral vector to the intestinal epithelium.

Among the pathologies liable to be prevented or treated by the compositions of the invention, there may be mentioned mainly: infectious mononucleosis, endemic Burkitt lymphoma, undifferentiated carcinoma of the nasopharynx (NPC), lymphomas appearing in immunocompromised patients, such as individuals who have undergone an organ transplant or even patients suffering from AIDS, or even certain types of Hodgkin lymphoma where EBV is involved. The subject of the invention is also a process for obtaining the recombinant vectors described above which comprises, after the actual construction step of these vectors by introducing one or more sequence (s) of foreign DNA into their genome , a step of transforming transformable cell lines of higher eukaryotes (in particular of human or animal origin) themselves comprising a distinct DNA sequence capable of complementing the part of the genome of the adenovirus essential for the replication of the latter and of which the abovementioned vector is devoid, said distinct sequence preferably being incorporated into the genome of the cells of said cell line.

As a preferred example of such cell lines, mention will be made of line 293 (Graham et al., 1977), a human embryonic kidney line which contains, integrated into its genome, the first eleven percent of the left end of the genome d adenovirus type 5 (Ad5). These make it possible to complement defective recombinant viruses which carry deletions from this region. Such a production process is more particularly described in European patent application No. 0 185 573 of 20/11/85.

After transformation of these cell lines, the vectors which have thus multiplied are recovered and purified.

The present invention will be described in more detail in the following description of the construction of a recombinant vector adenovirus comprising the gene coding for the glycoproteins gp340 / 220.

I. METHODS a) Cells and viruses The human embryonic renal cell line 293 transformed with Ad-5 (Graham et al., 1977), was used for DNA transfection as well as for the multiplication and titration of Ad. In fact, the cell line 293 complements the functions of the E1A and E1B genes and therefore allows replication of the defective Ad. For the construction of the recombinant Ad, human Ad5-dl324, carrying deletions in the E1 (3.9-10.5 mu) and E3 (78.5-84.3 mu) regions, was used (Shenk and Williams, 1984). Cell lines 293, Hela and Vero were maintained in an Eagle minimum essential culture medium with 10% fetal calf serum. b) Plasmids

The eukaryotic expression vector pMLP10 was described in the article by Levrero et al., 1991.

Plasmids pL1C and pL2C contain the major late promoter Ad2 (MLP) joined to its leader sequence

tripartite (leading sequence), followed by the region coding for the EBV gp340 and 220 glycoproteins and its polyadenylation signal or by the sequence originating from a complementary DNA clone (cDNA) of a spliced version of the 'MRNA, corresponding to the region encoding only for gp220. In addition, the constructs pL1CD and pL2CD were obtained from the preceding plasmids, after suppression of the DNA regions corresponding to the transmembrane and cytoplasmic domains of the proteins and addition of a stop codon and a polyadenylation signal from the gene coding for the SV40 virus early antigen (Tosoni-Pittoni et al.,

1989). c) DNA transfection and isolation of recombinant viruses The recombinant adenoviruses were obtained by recombination in vivo. Viral DNA fragments as well as linearized plasmids were transfected into 293 cells using the calcium phosphate precipitation method (Graham and Van Der Eb, 1973). Lysis plaques were isolated 10 days later, and the virus was amplified in culture. Viral DNA was extracted by the Hirt procedure (Graham et al., 1977) and the recombinant viruses were identified, mapped with restriction enzymes. d) Identification and guantification of EBV qp340 / 220

immunofluorescence (IF): indirect membrane immunofluorescence was carried out on the Hela or Vero cell lines 24 hours after infection with the recombinant viruses (Ad-gp340, Ad-gp220) or with Ad-dl324 as a negative control. In summary, the cells underwent trypsin treatment and were resuspended for 2 h at the concentration of 10 ⁶ cells per ml in a medium not comprising serum. 2x10 ⁶ cells were recovered and resuspended in 500 μl of anti-gp340 / 220 monoclonal antibody (New England Nuclear) diluted 100 times in phosphate buffer for 1 h at 37 ° C. An anti-mouse IgG antibody conjugated to fluorescein was added and the cells were incubated for 1 hour at 37 ° C. The washed cells were examined using a fluorescence microscope.

- immunoprecipitation: this technique was described for the isolation of gp340 / 220 from the culture medium in the article by Tosoni-Pittoni et al., 1989. The extracts of radiolabelled cells were obtained from 10 ⁷ infected cells exploded by sonication. e) detection of anti gp340 / 220 antibodies

- immunofluorescence: series of dilution of the sera were analyzed by IF on 293 cells fixed in an acetone / methanol mixture (1: 1), and previously transfected with a plasmid expressing gp340 / 220 (PLIC) in order to determine the levels in anti-gp340 / 220 antibody (Tosoni-Pittoni et al., 1989).

- neutralization experiment: this experiment was carried out according to the technique of the abortive transformation of lymphoid cells with EBV (De Schryver et al., 1974). f) Inoculation of animals with recombinant adenoviruses

- Inoculation of rabbits

In order to be inoculated, the adenoviruses were first purified by two passages in cesium chloride density gradients, and were dialyzed and diluted in PBS before inoculation. New Zeland rabbits were inoculated by different routes with 10 ¹⁰ pfu (assuming that a unit of optical density at 260 nm obtained for the viral suspension is equivalent to 3 × 10 ¹⁰ pfu / ml) of recombinant Ad or of Ad -dl324 as a negative control.

- Inoculation of cottontop tamarins

Six animals were used, which it had previously been verified that they did not have antibodies directed against gp340 / 220 of EBV by ELISA, against late EBV antigens (VCA) by immunofluorescence and finally against Adenovirus capsid proteins by immunofluorescence. Recombinant Ad expressing gp340 / 220 (Ad-gp340) was used to immunize four cottontop tamarins. An animal (R155 in Figure 4) was vaccinated with a non-recombinant control Ad (strain Ad-dl324) and another animal was not immunized (B159). The animals were vaccinated by three intramuscular injections. The first dose was 5.10 ⁹ pfu in one ml of buffered solution, and since the tamarins did not show any adverse reactions, the next two doses were 10 ¹⁰ pfu and 2.10 ¹⁰ pfu respectively in the 5th and 13th weeks after the first injection. The titers of the antibodies directed against the proteins of the capsid of the adenovirus and against the gp340 of the EBV were measured weekly. The tamarind plasma samples are tested for the antibody response against gp340 by ELISA and for the antibody response against the capsid antigens of the adenovirus by immunofluorescence.

II. RESULTS a) Construction of the recombinant adenoviruses

The adenoviruses containing the EBV gene gp340 / 220 under the control of MLP Ad2 were constructed as illustrated in FIG. 1. In order to obtain the plasmids of series IX, a BglII-HindIII fragment (nucleotides 3329 to 6242) of Ad5 was cloned between the BamHI and HindIII sites of the plasmids pLIC, pL2C or SphI (followed by repair of the DNA ends with Klenow polymerase) and HindIII of the plasmids pL1CD and pL2CD. This 3 kbp sequence contains the complete protein IX gene which is necessary to obtain viral DNA up to 97% of its normal size (Ghosh-Choudhury et al., 1987) and allows recombination to be carried out in vivo in cells. The reconstruction of recombinant Ad is described in the legend of Figure 1. b) Expression of the qp340 / 220 glycoproteins by recombinant adenoviruses

The capacity of the recombinant adenoviruses to express the gp340 / 220 glycoproteins was tested on various cell lines. Cells 293, Hela and Vero were infected with 1, 10, 100, 1000 pfu / cell, and recovered daily up to 36 h, 4 days or 7 days after transfection respectively. The strongest expression of gp340 / 220 was observed with the Hela cells 3 days after infection (FIG. 2B, well 5). The EBV-derived membrane proteins produced by the recombinants Ad-gp340 and Ad-220 were labeled with ³⁵ S methionine (FIG. 2A, well 1,2), are correctly glycosylated and could be detected using the surface of human cells infected by membrane immunofluorescence with different monoclonal antibodies (Figure 3). Similarly, the secreted forms of these proteins could be detected by immunoprecipitation from cell culture media in which the recombinants Ad-gp340D and Ad-gp220D were used for the infection (FIG. 2A, well 3.4 ; Figure 2B, wells 1, 4 and 5). On the other hand, the immunoprecipitation of human cell extracts in this case only shows that the truncated gp340 / 220 glycosylation precursors (FIG. 2B, well 3). After infection, the cells continue to secrete recombinant glycoproteins which can accumulate in the culture medium without undergoing apparent proteolysis (FIG. 2B, wells 4 and 5). c) Antibody response in rabbits inoculated with recombinant adenoviruses expressing EBV gp340 / 220 (Table 1)

Antibody responses directed against gp340 / 220 were followed in rabbits after injection of recombinant Ad. The immunization schedules are summarized in Table 1. After intravenous, intranasal, or intramuscular inoculation, a first dose of highly purified Ad-gp340 or Ad-gp220 recombinant virus (expressing membrane glycoproteins), anti- gp340 / 220 were detected two weeks later in all animals except one rabbit (rabbit # 33). After a second injection, a strengthening effect was observed on the animals, which then show a strong antibody response a week later. Relatively high titers were detected 30 weeks after the last inoculation. These antibodies can recognize the cells expressing gp340 / 220 by immunofluorescence and the proteins gp340 / 220 by immunoprecipitation (FIG. 2 A) and by immunoblotting according to the Western technique. There is no apparent difference between gp340 and gp220 in terms of their ability to induce specific antibodies.

The anti-gp340 / 220 titers obtained by rabbit inoculation with the recombinant Ad-gp340D (expressing the glycoproteins in secreted form) are much lower, and a long-term response could not be observed.

Different routes of inoculation have been tested. Intramuscular and intravenous injections are as effective as each other. The intranasal introduction of Ad triggers a slow first response, but similar antibody titers were obtained after the second vaccination (rabbit 68). The anti-gp340 / 220 positive sera from immunized rabbits have all been shown to be able to strongly neutralize the virus (see last column of Table 1). d) Antibody response in tamarins inoculated with recombinant adenoviruses expressing EBV gp340 / 220

Three weeks after the second injection, the 4 animals which received the recombinant virus develop antibodies against gp340 with titers ranging from 1/100 to 1/280. The titer of these antibodies reaches values of 1/440 to 1/1520 three weeks after the third injection. The 5 animals inoculated with adenovirus (including that which received the parental virus), develop antibodies against the capsid antigens of the virus between 1/80 and 1/640 after the 2nd injection and 1/640 to 1/280 after the third injection. No specific antibody is detected in the non-immunized animal. e) Protection of tamarin monkeys against lymphomas induced by EBV

Three weeks after the third injection, the 6 tamarins (including the non-immune control animal) received an intraperitoneal injection with a 100% tumorigenic dose of EBV (within 3 weeks) from '' a virus stock prepared from the B95 / 8 cell line. The 6 animals were then regularly examined by external palpation and measurement of the size of the lymph nodes and of the tumors thus induced.

The animal R155 which received the parental virus very quickly develops the disease with implication in the both peripheral and abdominal lymph nodes.

The animal had to be sacrificed on the 25th day after the injection of EBV. The unimmunized animal (B159) also develops the same symptoms. In contrast, the four tamarins immunized with recombinant Ad expressing gp340 are clearly protected and do not develop the disease after the injection of EBV. Only a few lymph nodes were transiently identified as swollen, 3 mm by 3 mm in diameter compared to 14 mm by 12 mm in diameter for unprotected animals. In addition, the tissues obtained after biopsy do not have tumor characteristics as in control animals.

III. RESULTS ANALYSIS

The inventors have demonstrated the expression of foreign genes in numerous cell lines infected with the aid of recombinant adenoviruses. In particular, the secreted form of gp340 / 220 of EBV could be easily purified from a medium containing no serum. This suggests the presence of a highly efficient post-translational transformation system secreting these polypeptide sequences. In addition, during infection, cells secrete glycoproteins which then accumulate in the cell environment. This provides a good source of antigens for the manufacture of a vaccine without risking contamination with EBV DNA. In the constructions of the present invention, the sequences coding for the antigens used for immunization or coding for the cytokines are placed under the control of the late major promoter (ubiquitous promoter) of Ad2, followed by the entire tripartite leader sequence; these DNA sequences are cloned into the E1 region of the virus. Indeed, mRNAs containing only the first exon of the leader sequence are translated 20 times less efficiently (Thummell et al., 1983; Davis et al., 1985). Recombinant adenoviruses recently described for the in vivo expression of foreign genes use a cloning site in the E3 region without deletion of the E1 gene (Morin et al., 1987; Johnson et al., Schneider et al., 1989).

The use of recombinant live viruses as vaccine vectors has many advantages over a subunit vaccine. These advantages include the possibility of producing viral glycoproteins in the absence of EVB DNA, obtaining correct post-translational modifications and an appropriate presentation of the antigen to effector T cells, a broad spectrum of immune responses, induction of long-term immunological memory, and finally a low cost of production and ease of inoculation of the vaccine (particularly in the case of use of a gastro-encapsulated vaccine). But the main drawback of using live recombinant viruses is the broad host spectrum and the possibility of horizontal transmission between humans and other species. In the case of vaccinia and polio virus, transmission to immunocompromised individuals through contact with vaccinated individuals has serious consequences.

The present invention demonstrates that a recombinant Ad5 having a deletion in the E1 region allows the expression in vivo of products of foreign genes and can induce the formation of neutralizing antibodies in rabbits against these products. The inventors have issued the hypothesis that insertion into the E1 region could have several advantages, in particular the fact of preventing the virus from replicating in vivo in. permissive hosts, in particular in humans, and consequently of limiting the risks of dissemination of the recombinant viruses, the latter then being able to carry out only a single cycle of infection in animal cells. Another advantage could be that of preventing a strong immunization against the proteins of the Ad capsid and of allowing multiple vaccinations without affecting the quality of the immunization against the foreign antigens expressed by this virus.

These different aspects are particularly important in the field of safety and for the potential development of recombinant Ad as a vaccine.

The differences found in the anti-gp340 / 220 titers between the membrane and secreted forms of gp340 / 220 can be explained by the different presentations of antigens to T cells depending on the fact that the antigen is on the surface of cells presenting the antigen or secreted. It has already been shown that the addition of an anchor sequence leads to a marked increase in the levels of antibodies recognizing a foreign antigen (Langford et al., 1986). The total absence of antibody response when using the secreted form of gp220 is more surprising. Truncated gp220 must be less stable in vivo, but it is even more problematic if the conformation adopted by this recombinant protein when the anchor domain has been deleted is weakly immunogenic in rabbits.

Another hypothesis would be that the single domain of gp340 which contains three repeats of an amphopathic motif of 21 amino acids (Beisel et al., 1985) could be important in triggering the antibody response, particularly when proteins are in secreted form.

The anti-gp340 / 220 positive sera obtained from immunized rabbits are all highly capable of neutralizing EBV. The anti-gp340 / 220 sera directed against the recombinant Ad expressing the secreted gp340 / 220 also neutralize EBV in vitro despite much lower titers. These results correspond to the fact that the secreted gp220 and truncated forms of gp220 in the C-terminal part, are still capable of binding to the EBV receptor (CR2) on the surface of B lymphocytes (Tanner et al., 1988). More precise mutations are needed inside the N-terminus of gp340 / 220 in order to identify the binding domain for CR2. Nevertheless, this work constitutes a preliminary step towards the identification of the gp340 / 220 epitopes involved in the immune response in terms of binding of antibodies with gp340 and neutralization of the virus.

The antibody response has been demonstrated to be able to persist appreciably for six months in rabbits. It is important to get long-term protection after vaccination.

As an antibody response neutralizing EBV in vivo has been obtained in rabbits vaccinated using recombinant Ad producing the membrane proteins gp340 / 220, the recombinants Ad-gp340 / 220 have been successfully tested for its ability to protect cottontop tamarins from EBV-induced lymphomas (see Figure 4).

The possibility of delivering adenoviruses via several inoculation routes including the intramuscular, intranasal, intraperitoneal routes, subcutaneous or oral, makes these adenoviruses effective tools for the in vivo expression of foreign genes. The possibility of obtaining such an effective immune response by intranasal introduction of the virus makes the latter very promising from the point of view of vaccine development. Adenoviruses have a particular affinity for the gastrointestinal wall and upper respiratory tract, for the tonsils and salivary glands in humans and some vertebrates. The use of recombinant Ad is therefore particularly advantageous for vaccination against EBV which replicates at the aforementioned sites, particularly by inducing the secretion of antibodies capable of protecting the oropharyngeal epithelium.

Table 1

Antibody titer (a)

Animal type virus Ways week after the 1st injection Neutralization

0 4 7 25 40 of the EBV (b)

Ad-gp340 30 iv - 16 256 512 S +++

31 iv - 512 1024 256 256

32 iv - 128 256 256 S

33 iv - - - 128 S

34 iv - 512 1024 S

35 iv - 512 2048 256 S

36 im - 1024 1024 256 256

38 im - 1024 2048 S

63 in - 32,512 S

Ad-gp220 40 iv - 1024 1024 128 256 +++ / ++

41 iv - 2048 1024 64 64

42 iv - 2048 1024 128 S

43 iv - 256 128 - S

44 iv - 2048 512 256 64

45 iv - 512 256 256 128

46 iv - 1024 512 16 S

47 iv - 1024 1024 256 S

48 im - 512 256 128 S

49 i m - 1024 512 256 4

Ad-gp340D 54 iv - 128 16 - S ++

55 iv - 256 - - S

56 iv - 512 - -

57 S

iv - 64 - -

58 S

iv - 8 16 - S

59 iv - 64 8 -

60 S

im - 256 8 -

61 S

im - 64 32 8 S

Ad-gp220D 65-67 iv - - - ND S ND

Ad dI324 80 iv - - - S - LEGEND OF FIGURES AND TABLE 1

Figure 1: Construction of recombinant adenoviruses

The DNA sequence coding for gp340 / 220 was inserted into the expression vector pMLP10. This plasmid comprises 450 nucleotides from the left end of the Ad5 genome (that is to say the inverted terminal repeat containing the origin of viral replication, the sequence necessary for the packaging of the virus and the activator sequence of the region E1A) followed by the sequence comprising the major late promoter of Ad2 flanked by the tripartite leader sequence. The plasmids pLCIX, pL2CIX, pLCDIX and pL2CDIX contain the different sequences allowing the expression of the various forms of gp340 / 220 described in the "methods" and "results" sections.

Recombinant adenoviruses are obtained by homologous recombination in vivo between the large fragment of the viral genome obtained after cleavage by a restriction enzyme and the homologous sequence existing in the plasmids described above (9.4 - 17% of the genome). The DNA mixture comprising the viral genome fragment (2.6 to 100% of the genome) purified after cleavage by the restriction enzyme Cla I and the plasmid linearized by the same restriction enzyme is transfected into 293 cells. Recombinant viruses respectively named Ad-gp340, Ad-gp220, Ad-gp340D and Ad-gp220D are then isolated.

Frames: viral sequences (white: viral genome; hatched: glycoprotein gene; black: SV40 sequences).

Arrow frame: MLP promoter; fine line: bacterial sequence. D / A: donor and acceptor splicing sites. 1 mu = 1% of the viral genome = 360 base pairs.

Figure 2: Detection of gp340 / 2220 by immunoprecipitation.

Polyacrylamide gel showing the gp340 / 220 glycoproteins immunoprecipitated from cells infected with recombinant Ad titrating to 1000 pfu (plaque forming unit) per cell. The radiolabelled proteins were extracted from 2.10 ⁶ cells or from the culture medium corresponding to the same number of cells for the secreted forms.

A: proteins detected 48 h after infection of the Hela cells with a positive rabbit polyclonal serum (well 1-4) or the preimmune serum (well 5), either from cell extracts (wells 1 and 2) or from the medium of culture concentrated in wells 3 to 5. The viruses used for the infection of the cells are: Ad-gp340, 2: Ad-gp220, 3: Ad-gp340, 4-5: Ad-gp220D.

B: gp 220 protein detected 48 h (well 4) or 72 h (other well) after infection of Vero (1-3) or Hela cells (4 and 5) with an anti-gp340 / 220 monoclonal antibody from the culture medium (wells 1, 2, 4 and 5) or cell extracts (well 3).

Figure 3: Membrane immunofluorescence.

Vero cells are infected with the recombinant virus Adgp220 at 10 p.p. / cell and treated with a monoclonal antibody antigp340 / 220 and an anti-fluorescent mouse anti-IgG.

Figure 4: Protection in cottontop tamarin monkeys after vaccination.

Three weeks after the third injection (week 16), the animals are tested with a 100% lymphomagenic dose (10 ^5.3 transforming units), leading to â the appearance of tumors within 21 days. The graph shown in the figure is obtained by plotting the tumor index of each animal (which represents the sum of the diameters of the palpable lymph nodes and the detectable tumor nodules in mm) against the time in days after the injection of EBV. White circles: animal immunized by the parental virus, sacrificed 25 days after the start of the experiment; white squares: non-immune animal.

Table 1: Antibody response in rabbits inoculated with recombinant Ad.

(a): titers of anti-gp340 antibodies detected by immunofluorescence. The titer of the sera is expressed as the inverse of the last positive dilution detected.

(b): the neutralization test is based on the abortive transformation of lymphoid cells with EBV. (-): negative result. (S): animal sacrificed or died during the experimentation period.

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Claims

1. Recombinant vector characterized in that it comprises, on the one hand, a genomic sequence of a defective adenovirus, that is to say devoid of the sequences necessary for its replication, and, on the other hand, an insert comprising a or several DNA sequences coding for any protein characteristic of EBV which is capable of inducing in an individual the formation of neutralizing antibodies protective against EBV and, where appropriate of protective cells, in particular cytotoxic T cells and T helper, this insert being placed under the control of a promoter present or previously inserted into the genome of the adenovirus.

2. Recombinant vector according to claim 1, characterized in that its genome comprises a DNA sequence coding for the glycoprotein gp340 or gp220 of the Epstein-Barr virus (EBV), or for any fragment of these proteins which is capable of inducing the formation of protective antibodies against EBV in an individual, and, where appropriate, protective cells, in particular cytotoxic T cells and T "helper" cells.

3. Recombinant vector according to claim 1 or claim 2, characterized in that its genome comprises a DNA sequence coding for an EBV membrane antigen involved in the mechanism of fusion of the EBV viral envelope with the membrane of B lymphocytes and epithelial cells, and more particularly the glycoprotein gp85, or for any fragment of this protein capable of inducing the formation of said protective antibodies or protective cells.

4. Recombinant vector according to one of claims 1 to 3, characterized in that the genomic sequence of the defective adenovirus nevertheless comprises those of the sequences which in this genome are the carrier of the genetic information necessary for the corresponding adenovirus to penetrate into the cells which can be infected with it, and, where appropriate, all of the essential sequences necessary for the packaging of this adenovirus.

5. Recombinant vector according to one of claims 1 to 4, characterized in that the genome of the non-replicable or defective adenovirus is devoid of the nucleotide sequences of the E1 region necessary for the replication of this adenovirus.

6. Recombinant vector according to one of claims 1 to 5, characterized in that the adenovirus genome also comprises a DNA sequence coding for a protein stimulating the immune system.

7. Recombinant vector according to claim 6, characterized in that the adenovirus genome comprises an insertion nucleic acid coding for one or more interleukins chosen from interleukins-2, -3, -4, -5, -6 , -7 or any fragment thereof which may stimulate the immune system.

8. Pharmaceutical composition comprising one or more recombinant vector (s) according to one of claims 1 to 7, in combination with a pharmaceutically acceptable vehicle.

9. A vaccine directed against pathologies caused by EBV in humans or animals, this vaccinating composition comprising one or more recombinant vector (s) according to one of claims 1 to 7, in combination with a pharmaceutically acceptable vehicle.

10. Method for preventing or treating pathologies caused by EBV in humans or animals, this method comprising the administration in humans or animals likely to be infected with EBV or likely to be infected, of a vaccine or a pharmaceutical composition according to claim 9 or claim 8 respectively.

11. Method according to claim 10 characterized in that it allows the prevention or treatment of infectious mononucleosis, endemic Burkitt lymphoma, undifferentiated carcinoma of the nasopharynx (NPC), lymphomas appearing in immunocompromised patients, such as individuals having undergone an organ transplant or patients suffering from AIDS, and certain types of Hodgkin lymphoma.