MXPA95003231A - Immortalized lymphocytes for the production of vi-free propelles - Google Patents

Abstract

The present invention is directed to the construction of recombinant plasmids containing the genetic information of the Epstein-Barr virus ("EBV"), involved in the immortalization of human juvenile cells, but lacking one or more genes involved in the lithic replication of EBV. The recombinant plasmids have the advantage of being able to immortalize human lymphocytes, while being able to generate infectious particles of EBV. The invention also provides human lymphocytes that have been successfully infected to immortalized by the recombinant plasmids. These human lymphocyte clones can be used as cell factories to produce desired proteins and as distribution vehicles for gene therapy. Furthermore, the invention establishes a method by means of which the desired proteins can be produced in immortalized cell factories, without the generation of infectious EBV particles. These proteins can be the normal cell products of immortalized human lymphocytes, or they can be proteins encoded by foreign genes, which have been cloned into the lymphocytes.

Description

"IMMORTALIZED LYMPHOCYTES FOR THE PRODUCTION OF VIRUS FREE PROTEINS" Inventors and Applicants: WILLIAM M. SUGDEN, North American, domiciled at 2130 Fox Avenue, Madison, Wisconsin 53711, E.U.A. and WOLFGANG F. HAMMERSCHMIDT, German, domiciled at Schraemel Strasse 4, D-81377 Munich, Germany.

BACKGROUND OF THE INVENTION TO . Field of the Invention This invention relates to the technology of Recombinant DNA It provides recombinant plasmids derived from Epstein-Barr virus ("EBV"), which can immortalize human lymphocytes, while making them incapable of producing infectious viral particles. The immortalized lymphocyte clones can then be used as cell factories to produce virus-free proteins, as delivery vehicles for human gene therapy.

B. Previous Technique The last decade has witnessed progress in the development and application of recombinant DNA techniques in eukaryotes. For example, re-troviral vectors have been developed to possess foreign genes within certain eukaryotic hosts. Progress has also been made in the development of vector systems suitable for use with human B lymphocytes. Apart from its applications in pure research, these advances have potential therapeutic applications. But more progress is needed. In particular, it is desirable to develop vectors that can immortalize human lymphocytes. Such immortalized cell lines could be used as cell factories for the production of desired proteins, and as delivery vehicles for human gene therapy. EBV has the ability to transform human lymphocytes into cell lines that proliferate indefinitely in culture. Because EBV has latent and lytic replication cycles, however, human lymphocytes immortalized by EBV can produce infectious EBV particles provided that latent, resident EBV enters lytic replication. This risk of generation of infectious particles makes the cells immortalized by EBV, not suitable for therapeutic applications. There is thus a need to develop recombinant plasmids containing the EBV genetic information involved in the immortalization of human lymphocytes, but lacking one or more genes required for the lytic replication of EBV. And there is an additional need to use such plasmids, to produce immortalized human lymphocytes, which can generate desired proteins that are not contaminated by infectious EBV particles.

BRIEF DESCRIPTION OF THE INVENTION This invention is directed to a recombinant plasmid, to human lymphocytes immortalized by the recombinant plasmid, and to a method for the production of virus-free proteins, useful in various therapeutic applications. The recombinant plasmid incorporates viral DNA that can immortalize human lymphocytes without allowing the lytic replication of the virus of origin. The recombinant plasmid is formed by cloning selected DNA segments of Epstein-Barr virus ("EBV") on a prokaryotic plasmid backbone. The recombinant plasmid includes genes and elements that act in the cis position, which are involved in the immortalization of human lymphocytes or in the latent replication of EBV. The recombinant plasmid also lacks one or more genes involved in the lytic replication of EBV. Consequently, the plasmid retains the ability to immortalize human lymphocytes, while lacking the ability to enter the lytic replication of EBV, and thus to produce infectious EBV particles. The plasmid is thus a useful vector for the transformation of human lymphocytes and for the introduction of foreign genes coding for desired products.

A second aspect of the invention provides human lymphocytes that have been successfully infected and immortalized by the recombinant plasmid of the invention. Since clones of human lymphocytes infected by the plasmid lack some of the genetic information involved in the lithic replication of EBV, the clones are inherently incapable of generating and releasing infectious EBV particles. Human lymphocyte clones can therefore be used as cellular factories for the production of desired proteins and as delivery vehicles for human gene therapy. A third aspect of the invention relates to a method for the production of desired proteins, which are not contaminated by the infective particles of EBV. The method has several variations. In one variation, immortalized human lymphocytes are simply used as cellular factories to produce protein products, either antibodies or lymphokines. To produce antibodies of a desired specificity, B lymphocytes must be obtained from an antigenically challenged or diseased person. The resulting B cell clones are then screened using well-established immunological techniques, to identify clones that produce antibodies with desired specificities. These antibodies can then be used to promote passive immunity.

Yet another variation of the method involves the cloning of a gene encoding a desired protein product - within the recombinant plasmid described in this invention, or within the genome of human lymphocyte clones. Human lymphocytes immortalized by successful infection with the recombinant plasmid could produce the encoded protein without the generation of infectious EBV particles. A third variation of the method described in this invention involves the use of immortalized lymphocyte clones, as vehicles for human gene therapy. This method includes the injection of immortalized cells into human containers. Immortalized human lymphocytes could then produce desired proteins in situ, regardless of whether the proteins are antibodies, lymphokines or the product of a foreign gene that was cloned into the plasmid. Since human lymphocytes could typically be extracted from the same person who could later receive the lymphocyte clones, complications resulting from immunological recognition by the recipient of the injected cells could be avoided.

DETAILED DESCRIPTION OF THE INVENTION General description This invention encompasses a recombinant plasmid containing the genetic information of EBV involved in the immortalization of human lymphocytes, but lacking one or more genes involved in the lytic replication of EBV. Since the recombinant plasmid is unable to perform lytic replication, it can not produce infectious EBV particles. The invention also comprises clones of immortalized lymphocytes, which have been successfully infected by the recombinant plasmid of the invention. The invention further provides a method for the production of the desired protein, which is free of infectious EBV particles.

The Recombinant Plasmid The invention provides a recombinant plasmid containing EBV genes and elements that act in the cis position, involved in the immortalization of human lymphocytes and in the latent replication of the plasmid. The recombinant plasmid lacks one or more EBV genes involved in the lytic replication of EBV. Since the recombinant plasmid contains only a portion of the EBV genome, it is called a mini-EBV. The EBV genes of lymphocyte immortalization, which are included in the recombinant plasmid, are LMP1, EBNA2, EBNA3a, EBNA3c, and EBNA1. For the description and additional details of these genes, see Bettina Kempkes et al., Immortalization of Human B Lym-phocytes by a Plasmid Containing 71 Kilobase Pairs of Epstein-Barr Virus DNA, 61 J. of Virol. 231-38 (January 1995) (hereinafter "Kempkes et al."); see also Paul J. Farrell, Tumorigenic DNA Viruses, in 8 Advances in Viral Oncology 103-27 (George Klein ed., 1989); Elliot Kieff & David Liebowitz, Epstein-Barr Virus and Its Replication, in 2 Fields Virology 1889-1920 (Bernard N. Fields et al., Eds., 2nd ed., 1990). Each of these articles is incorporated by reference herein. The LMP2a, LMP2b, EBER1, EBER2, EBNALP and EBNA3b genes can also be included in the recombinant plasmid, but human lymphocytes can be immortalized without them. The elements that act in the cis position, present in the recombinant plasmid are TR, oriP, and oriLyt. OriLyt allows the recombinant plasmid to be packaged, with the help of helper cells, into viral particles that can more easily infect human lymphocytes. Kempkes et al., Cited above, identify and describe these genetic elements, and that description is incorporated by reference herein. The recombinant plasmid also lacks one or more of the genes that are involved in the lytic replication of EBV. The genes are BALF5, BALF2, BMRF1, BSLF1, BBLF, BBLF 2/3, BcLFl, BZLF1, BRLF1, and BMLF1. The absence of any of these genes results in a recombinant plasmid that is inherently incapable of performing lytic replication. Elizabeth D. Fixman and collaborators, Trans-Acting Requirements for Replication of Epstein-Barr Virus OriLyt, 66 J. of Virol. 5030 (August 1992) describe these genes in detail-including their functions and positions, and this description is incorporated by reference herein. The various EBV genes discussed above, the presence or absence of which partially defines the invention, have been discussed in the prior art and are well characterized. See in general id.; Paul J. Farrell, Tumorige-nic DNA Viruses, in 8 Advances in Viral Oncology 103-27 (George Klein ed., 1989); Elliot Kieff & David Liebowitz, Epstein-Barr Virus and Its Replication, in 2 Fields Virology 1889-1920 (Bernard N. Fields et al., Eds., 2nd ed., 1990) (each of which is incorporated by reference herein).

A _; _ Preparation of the Recombinant Plasmid All materials used in the construction of the recombinant plasmid are commercially available, unless otherwise specified. The recombinant plasmid is constructed by cloning the EBV genes and cis-acting elements, involved in the latent replication and immortalization of human lymphocytes, on a vertebral column of the prokaryotic plasmid, using well-known cloning techniques . The backbone of the prokaryotic plasmid comprises a prokaryotic plasmid that has been stripped of all functionally significant genes, with the remaining DNA that serves as a receptacle for other DNA sequences, which are cloned on the prokaryotic plasmid. A cloning technique called the multi-step chromosome construct is especially preferred because it allows large but deliberately selected EBV segments to be added to the prokaryotic backbone. For a detailed discussion of this technique, see O'Connor et al., Construction of Large DNA Segments in Escherichia coli, 244 Science 1307-1312, which is incorporated by reference herein. The technique involves the sequential addition to an initial plasmid of partially overlapping DNA segments, each of which is added via a different shuttle vector plasmid. See Id. and Kempkes et al., 61 J. of Virol. on pages 232-33, which are incorporated by reference herein. The chromosome construction technique thus allows the step-by-step construction of a recombinant plasmid with a defined composition. Using the preferred chromosomal construct technique, the recombinant plasmid is constructed by cloning large but deliberately selected regions of the EBV genome within an E. coli-based plasmid of factor F. Nine plasmids from the competent strain B95-8 , immortalized, of EBV, are established in E. coli. Plasmid p931.12 of factor F serves as the container for homologous recombination in E. coli, with the following shuttle plasmid, namely p935.1. In the preferred embodiment, the plasmid p935.1 and all other recombinant plasmids are generated using the cloning vector pMB096. Homologous recombinations are carried out in recA + E. coli RVsmc or recA E. coli CBTS that possess a recA amber allele and a temperature sensitive amber suppressor. The intermediate plasmids are resolved through the use of a plasmid expression molecule via the two rfsF sites present in the cointegrate, and the prokaryotic F-factor backbone is conserved together with the recombined EBV insert. Partially overlapping plasmids are aggregated in consecutive steps, with each plasmid adding a segment of DNA that binds to the structure of the recombinant backbone, under development, through homologous recombination. The recombinant plasmid pl244.8a resulting from the preferred method includes the genes LMP1, LMP2a, LMP2b, EBER1, EBER2, EBNA-LP, EBNA2, EBNA3a, EBNA3b, EBNA3c, and EBNA1, and the elements acting in the cis position, TR, oriP and OriLyt. But plasmid pl244.8a lacks the genes BALF5, BALF2, BBLF 2/3 and BcLFl. Since each of these four genes is involved in the lytic replication of EBV, the plasmid pl244.8a is unable to perform the lytic replication, and is thus unable to produce infectious particles of EBV. Disabling lithic replication of EBV, however, could be achieved by omitting any group of one or more genes that are involved in the lithic replication of EBV. Recombinant plasmids capable of initiating and maintaining the immortalization of human lymphocytes without the aid of the helper virus can be created by rescue of the recombinant plasmid of the lymphocytic clones lacking any detectable trace of the helper virus. For more details see Kempkes and collaborators, 61 J. of Virol. on page 233, which is incorporated by reference herein. The ability of the rescued plasmids to initiate and maintain the immortalization of human lymphocytes can be tested by exposing the lymphocytes to serial dilutions of reserve viruses, plating the infected cells in semi-solid medium, and plotting the number of proliferating colonies as a function of serial dilutions of viral stocks. The resulting "kinetics of a shock" reveals whether a simple recombinant plasmid can effect immortalization without the aid of the cooperating virus. For more details see Kempkes et al., 61 J. of Virol. on page 233, which is incorporated by reference herein. B-cell clones lacking any trace of the P3HR1 helper virus can be studied by rescuing their respective mini-EBVs within E. coli, and sequencing them. The extensive analysis of these rescued plasmids reveals that their structures are the same as the original p244.8a recombinant plasmid, except at the EBNA3a locus. The altered plasmids are capable of initiating the immortalization of B cells, indicating that the EBNA3 gene of the sil-vestre type is required for such initiation. Because these recombinant plasmids possess the intrinsic ability to initiate, as well as maintain the immortalization of human lymphocytes, these are preferred. The wild-type EBNA3 gene was also inserted into pl244.8a by standard cloning techniques, and this procedure is especially preferred. In summary, any recombinant plasmid that incorporates the EBV genes that are involved in the initiation and maintenance of the immortality of human lymphocytes, but lacks one or more of the genes involved in the lytic replication of EBV, is within the present invention, notwithstanding the presence of any other genetic information. The invention is exemplified but not limited to the factor F plasmid system and the shuttle vectors used in the preferred embodiment. Virtually any prokaryotic plasmid can be used, as long as suitable shuttle plasmids are established.

B. Use of Recombinant Plasmid The recombinant plasmid provided by this invention can be used to immortalize human lymphocytes, as discussed below. The plasmid can also be used as a vector to carry foreign genes to human lymphocytes. The recombinant plasmid can also be used as a research tool to further elucidate the process of immortalization and the latent and lytic replication cycles of EBV.

Immortalized Human Lymphocytes A second aspect of the invention provides clones of human lymphocytes that have been infected and immortalized by the recombinant plasmid discussed above, and described in this invention. Since the clones lack some of the genetic information involved in the lithic replication of EBV, they are inherently incapable of generating and releasing infectious EBV particles. Human lymphocyte clones can therefore be used as cell factories for the production of desired proteins, and as delivery vehicles for human gene therapy.

Elaboration of Immortalized Human Lymphocytes Human lymphocytes are generally resistant to DNA uptake and expression. For ease of use, the recombinant plasmid is packaged or "encapsidated" within a viral particle, to infect human lymphocytes. The incorporation of the elements acting in the cis, oriLyt and TR position (terminal repeats) within the recombinant plasmid allows the amplification and subsequent packaging of the plasmid. OriLyt is the EBV origin for lithic replication. See Lytic Origin of Replication for Epstein-Barr Virus (EBV), US Patent No. 5,194,601, issued March 16, 1993, the description of which is incorporated by reference herein. The presence of oriLyt in the recombinant plasmid allows it to be amplified. The presence of TR elements (terminal repeats) allows the amplified plasmid to be excised and packaged into viral particles by lymphoblastoid cooperating cells containing the endogenous cooperating virus. For details, see W. Hammerschmidt and B. Sugden, 340 Nature on page 393, which is incorporated by reference herein. Once encapsidated, the recombinant plasmid can more easily infect human lymphocytes. A helper cell line is a clone of cells that is latently infected with a strain of non-immortalization of EBV, which has all the trans-acting genes needed to replicate and package the recombinant plasmid. See Kempkes et al., 61 J. of Virol. at pages 232-33, the description of which is incorporated by reference herein. In a preferred embodiment, a clone of het cells free of Burkitt's lymphatic het infected with P3HR1, called the helper cell clone HH514, is used. See L. Heston, New Epstein-Barr Virus Variants from Cellular Subclones of P3J-HR-1 Burkitt Lymphoma, 295 Nature 160-63 (1982), which is incorporated by reference herein. These helper cells HH514 are developed in medium RPMI supplemented with 10% fetal calf serum. The recombinant plasmid is introduced into the appropriate helper cells by electroporation or other appropriate technique. For further clarification, see Kempkes et al., 61 J. of Virol. on page 232-33, the description of which is incorporated by reference herein. The plasmid is preferably introduced together with the BZLF1 gene acting in trans, viral position, to induce the lytic phase of the EBV life cycle. After several days, the viral particles that have been released from the Used cells are harvested. Id. Each viral particle contains either the genomic DNA of the helper virus, the encapsidated copies of the recombinant plasmid, or the possible recombinations between the two. The reserve virus is filtered to eliminate the cells, and used to infect human lymphocytes: preferably human, primary B lymphocytes. B cells can be harvested from spinal cord blood, solid peripheral blood coating fractions from adult, or other well-known sources. After being exposed to the viral particles, the human lymphocytes are plated at limiting dilution on a layer of feeder cells of human fibroblasts, lethally irradiated. Immortalized lymphocyte clones develop from lymphocytes that were infected by the recombinant plasmid, alone or in conjunction with the helper virus, but not from the cells that were infected by the helper virus alone. To determine whether clones of the immortalized lymphocytes contain the recombinant plasmid alone or in combination with the DNA of the helper virus, standard analyzes of Southern staining and PCR are used to select the clones. For details concerning these well-known analyzes, see Kempkes, 61 J. of Virol. on pages 232-33, which is incorporated by reference herein. It can be found that the clones do not contain any detectable trace of helper viruses, with a level of sensitivity that uses PCR analysis of less than 5 DNA molecules of cooperating viruses per 30,000 cell genomes. The clones are stably immortalized. The non-encapsidated recombinant plasmid can also be introduced into B lymphocytes directly by electroporation, or some other suitable technique. The proliferation of B cell clones is raised in the culture after the introduction of naked plasmid DNA, but less efficiently than when the recombinant plasmid is packaged or encapsidated before being used to infect B cells. The inclusion of a step of packaging is thus preferred. Despite being injected, the recombinant plasmid replicates extrachromosomally within the infected lymphocytes, as expected. This can be demonstrated by applying the Gardella gel technique. See in general T. Gardella et al., Detection of Circular and Linear Herpesvirus DNA Molecules in Mammalian Cells by Gel Electrophoresis, 50 J. of Virol. 248-54 (1984), the description of which is incorporated by reference herein.

B. Use of Immortalized Human Lymphocytes The invention provides clones of human lymphocytes that have been successfully infected and immortalized by the recombinant plasmid. Although the clones are stably immortalized by the presence of EBV genes on the recombinant plasmid, the cells intrinsically lack the ability to enter the EBV lytic phase and thereby produce infectious EBV particles. Immortalized human lymphocytes are thus eminently suitable for the production of useful EBV-free proteins, and as delivery vehicles in human gene therapy.

Method of Production of Desired Proteins that are Free of Contamination by EBV A third aspect of the invention relates to a method for the production of desired proteins that are free of infectious EBV particles. The method involves several variations: (1) the production of a desired protein that is native to a human lymphocyte - usually an antibody or lymphokine, (2) the production of a desired protein that is encoded by a foreign gene that has been introduced in a human lymphocyte, and (3) the production of a desired protein - either native or foreign - in which the immortalized host lymphocyte is effectively inserted into a human container, so that the protein is produced and distributed in situ.

All three variations include the infection of human lymphocytes with a recombinant plasmid containing the genetic information of EBV involved in the immortalization of human lymphocytes, but lacking one or more genes involved in the lytic replication of EBV. All three variations thus involve the construction of a recombinant plasmid according to the techniques discussed above, or other well-known cloning techniques. Alternatively, the recombinant plasmid can be obtained commercially. Notwithstanding being obtained, the recombinant plasmid is then introduced into helper cells by electroporation or some other appropriate technique, as discussed above, and the lytic phase of EBV replication is induced. After several days, the encapsidated EBV viral particles are harvested and can be used to infect human lymphocytes. The three variations of the method are also different in important aspects. The first variation involves the use of immortalized lymphocytes to produce large amounts of their common protein products, namely antibodies or lymphokines. In the case of antibodies, the method preferably demands that human B cells be extracted from antigenically challenged individuals, so that antibodies of the desired specificity can be produced. For example, to generate specific antibodies for HIV virus antigens, B cells could be extra-human from HlV-positive individuals. These B cells could then be immortalized through infection with the recombinant plasmid of the invention, as discussed above. The resulting immortalized B cell clones could then be screened, using well-known immunological techniques, to identify clones that produce antibodies specific for the relevant HIV epitopes. See E. Harlow and D. Lane, Antibodies on pages 139-241 (Cold Spring Harbor Laboratory, 1988), which is incorporated by reference herein. The desired B cell clones might have to be subjected to limiting dilutions and repeated analyzes of Southern blotting and PCR, to ensure complete absence of the HIV DNA. See id. To conduct these analyzes, DNA fragments derived from the HIV virus would have to be available. This variation of the method is generalizable: it provides a means for the production of monoclonal antibodies specific for the epitopes of virtually any well-known pathogen. The antibodies can then be used to establish passive immunity in human patients or for experimental purposes. The second variation of the method involves the cloning of a foreign gene encoding a desired protein product within a recombinant plasmid, previously described, or within the genome of the host lymphocyte. Any well-known cloning technique can be used to insert the desired gene. See J. Sambrook et al., Molecular Cloning (Cold Spring Harbor Laboratory, 1989), which is incorporated by reference herein. This variation of the method allows mass production by immortalized human lymphocytes of the protein encoded by the cloned gene. See Example 5 below, for more details. A third variant of the method involves the injection of immortalized human lymphocytes into human containers. Immortalized human lymphocytes can then produce the desired protein in situ, provided the product consists of antibodies, lymphokines, or the product of a cloned gene. Since the primary human lymphocytes employed in the method will generally be obtained from the same person who subsequently receives the clones of immortalized lymphocytes, immunological recognition by the recipient's immune system is avoided. For more details, see K. W. Culver, Gene Therapy, chapter 4 (Mary Ann Lie-bart, 1994), which is incorporated by reference in this; see also Example 6, infra. In this invention, recombinant plasmids containing all the EBV genetic elements involved in the immortalization of human lymphocytes, but lacking one or more genes essential for the lytic replication of EBV, were generated. These recombinant plasmids have the ability to initiate and maintain immortalization in human lymphocytes, however, they intrinsically lack the ability to enter the lytic replication cycle, and thus produce infectious EBV particles. The invention also provides immortalized human B lymphocytes that have been successfully infected with such recombinant plasmids. In addition, the invention provides a method for the production of a desired protein that is free of infectious EBV particles. The present invention is not limited to the particular species of recombinant plasmids described in the preferred embodiments. Rather, the invention comprises any recombinant plasmid containing the EBV genes necessary for the initiation and maintenance of immortalization, but lacking one or more genes necessary for lytic replication, notwithstanding the presence of genetic information. additional, or the spatial order of the genes contained in the recombinant plasmid. These observations and reservations - made with reference to the claimed recombinant plasmid - apply function to the immortalized human lymphocytes and to the methods also advanced in the appended claims.

Example 1 Construction of Plasmid p! 244.8a Plasmid pl244.8a was constructed by cloning large but deliberately selected portions of the EBV genome into a factor F plasmid using the chromosomal construct technique. Nine plasmids from the competent-immortalized EBV B95-8 strain were established in E. coli. Plasmid p931.12 of factor F served as the container for homologous recombination in E. coli, with the following shuttle plasmid, namely p935.1. Plasmid p935.1 and all other recombinant plasmids were generated using the cloning vector pMB096. Homologous recombinations were carried out in recA + E. coli RVsmc or recA E. coli CBTS possessing a recA amber allele and a temperature sensitive amber suppressor. The combined plasmids were resolved through the use of a ResD expression plasmid, via the two rfsF sites present in the cointegrate, the prokaryotic backbone of factor F is conserved together with the recombined EBV insert. Neighboring plasmids, which partially overlap, were added in consecutive steps. The aggregated plasmids were sequentially linked to the structure of the recombinant, growing spine through homologous recombination, and the resulting plasmids were then resolved. The pl244.8a plasmid that resulted at the end of the multi-step chromosomal construction technique is 83,851 base pairs in length, including the prokaryotic backbone and a marker gene which plays no role in the invention. Total aggregate EBV DNA reached 71 kbp of the EBV genome, which by itself is approximately 165 kbp in length. Plasmid pl244.8a includes the genes LMP1, LMP2a, LMP2b, EBER1, EBER2, EBNA-LP, EBNA2, EBNA3a, EBNA3b, EBNA3c, and EBNA1, and the elements acting in cis position, TR, oriP, and OriLyt. Plasmid pl244.8a lacks the BALF5, BALF2, BBLF 2/3 and BcLFl genes. The plasmid pl244.8a is thus inherently incapable of supporting the lytic replication of EBV. This, however, is capable of maintaining the immortality of B cells.

Example 2 Immortalization of Human B Lymphocytes Plasmid pl244.8a was constructed as detailed in Example 1, and also in the specification. Samples were acquired from helper cell clone HH514, which is a het-free cell clone of the Burkitt lymphoma cell line P3HR1. Ten micrograms of plasmid pl244.8a DNA was transiently introduced into HH514 cells by electroporation, together with 10 micrograms of pCMV-BZLF1, to induce the lytic life cycle. The HH514 helper cells were developed in RPMI medium supplemented with 10% fetal calf serum. The particles released from the virus were harvested 5 days later. The viral particles released from the lysed helper cells contained either the genomic DNA of the helper virus P3HR1, encapsidated copies of pl244.8a, or possible recombinations between them. This reserve virus was then sterilized, filtered, and used to infect human, primary B cells, derived from spinal cord blood or from adult peripheral blood tight coating fractions. The infected B cells were then plated at limiting dilution in plates with 96-well pools, on a layer of lethally irradiated human fibroblast feeder cells. Immortalized cell clones were developed from B cells that were infected by plasmid pl244.8a, but not from cells that were infected by the P3HR1 helper virus alone. These B cell clones have proven to be stably immortalized.

Example 3 Rescue of Plasmids p! 244.8a-6.6 and p! 244.8a-8.4 In initial experiments, seven of the forty-seven immortalized B cell clones were found to lack any detectable trace of P3HR1 helper virus. further, a clone that was doubly infected by the DNA of pl244.8a and P3HR1, gradually lost the DNA of the cooperating virus over time. These observations raised the possibility that pl244.8a would be able to initiate immortalization in the absence of P3HR1. Adult B lymphocytes were exposed to serial dilutions of virus stocks that included viral particles containing pl244.8a. The infected cells were plated in a semi-solid medium, and the number of proliferating colonies was plated as a function of the serial dilutions of the reserve virus. The results revealed the "kinetics of two shocks", indicating that a simple pl244.8a plasmid is incapable of initiating immortalization. The P3HR1 helper virus must accompany p244.8a to initiate the proliferation of B lymphocytes. Two of the B cell clones lacking detectable traces of the P3HR1 helper virus were further studied by rescue of their respective mini-EBVs, within E. coli and sequencing them. Extensive analyzes of these plasmids indicate that their structures are the same as the parental plasmid pl244.8a, except at the EBNA3 locus. The two altered plasmids were rescued from cell clones Be253.30 and Be253.33, and were designated pl244.8a-6.6 and pl244.8a-8.4, respectively. These altered plasmids proved capable of initiating and maintaining the immortalization of B cells, indicating that the wild-type EBNA3 gene is much more involved in such initiation. Plasmids capable of initiating and maintaining the immortality of B cells were also made by cloning a wild-type EBNA3 gene, into pl244.8a.

Example 4 Generation of Specific Antibodies for HIV Envelope Protein, Using Immortalized B Cells To generate immortalized B cells that secrete antibodies specific for the HIV envelope protein, B cells from an HIV-positive donor could be infected with the packaged recombinant plasmid of the invention. The infected cells could then be plated on microliter discs, on an irradiated feeder layer, under conditions of limiting dilutions. Each well with proliferating B cell clones could then be tested for antibodies specific to the HIV envelope protein, using ELISA or some other suitable immunological assay. Wells with clones that prove to be positive could be subcloned into microliter discs and tested again for the presence of antibodies with appropriate specificities. This process could be repeated until it was found that all of the progeny cloned from an identified well secreted antibodies to the HIV envelope protein. Useful clones are then selected by PCR to detect the presence of unwanted EBV lytic genes, and HIV. Only cells that are free of these contaminants are used later. The techniques cited in this example are well established. See E. Harlow and D. Lane, Antibodies on pages 139-241 (Cold Spring Harbor Laboratory, 1988), which is incorporated by reference herein.

Example 5 Cloning a Strange Gene into Human B Lymphocytes, Using p! 244.8a The tissue plasminogen activator gene (TPA) with its translational terminator could be cloned into one of the plasmid derivatives used to construct the recombinant plasmid. The TPA gene would have to be cloned behind an efficient promoter: the immediate early promoter of the human cytomegalovirus could function. A possible site within the recombinant plasmid that can host the expressed, foreign genes, falls between the open reading frames for EBNAl and LMP. Approximately 50 kbp of EBV DNA has been removed from this region in the recombinant plasmid, and no required transcription occurs in this region in the wild-type EBV. A plasmid derivative (e.g., from pl242.1) could be constructed, which contains and expresses TPA. This derivative could then be used in the multi-step chromosomal construction technique, so that the TPA gene could be incorporated within the region between EBNA-1 and LMP of the plasmid p244.8a. For a detailed description of the techniques used to construct and test a recogminating plasmid derivative containing a foreign gene, see J. Sambrook et al., Molecular Cloning (Cold Spring Harbor Laboratory, 1989), which is incorporated by reference herein. .

Example 6 Use of Human B Lymphocytes, Immortalized, as Vectors for Human Genetic Therapy B lymphocytes of a patient with a deficiency in adenine deaminase (ADA) could be infected with a recombinant plasmid within which the human gene for ADA is cloned and efficiently expressed, according to the techniques described in Example 5. The cells immortalized could be analyzed for ADA expression levels by testing them for protein and RNA, as described in J. Sambrook et al., Molecular Cloning (Cold Spring Harbor Laboratory, 1989), which is incorporated by reference herein. B cell clones lacking the helper virus and expressed levels of ADA expressed, could then be reintroduced into the patient to provide therapeutic levels of ADA in situ. For details, see K. W. Culver, Gene Therapy, chapter 4 (Mary Ann Liebart, 1994), which is incorporated by reference herein. These and other objects and advantages of the present invention are discussed, illustrated, and supported by the above brief description and detailed description, including examples and preferred embodiments, the embodiments, however, do not define the full scope of the invention. Rather, the invention is defined primarily by reference to the appended claims It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following:

Claims (10)

1. A recombinant plasmid, characterized in that it comprises: a prokaryotic plasmid backbone attached to a DNA segment comprising a group of EBV genes, including LMP1, EBNA2, EBNA3a, EBNA3c, and EBNAl, and a group of elements acting in the cis position of EBV, including TR, oriP, and oriLyt; wherein the recombinant plasmid is free of at least one EBV lytic gene selected from the group consisting of BALF5, BALF2, BMRF1, BSLF1, BBLF4, BBLF 2/3, BcLF1, BZLF1, BRLF1, and BMLF1, or combinations thereof .

2. A recombinant plasmid according to claim 1, characterized in that each EBV gene included is associated with its wild-type EBV promoter.

3. The recombinant plasmid according to claim 1, further characterized in that it comprises the EBV genes selected from the group including LMP2a, LMP2b, EBER1, EBER2, EBNA-LP, and EBNA3b, or combinations thereof.

4. Immortalized human lymphocytes, characterized in that they comprise: human lymphocytes that have been infected with a recombinant plasmid comprising: a prokaryotic plasmid backbone attached to a DNA segment comprising a group of EBV genes including LMP1, EBNA2, EBNA3a , EBNA3c, and EBNAl, and a group of the elements that act in the cis position, of EBV, which include TR, oriP, and oliLyt; wherein the recombinant plasmid is free of at least one EBV lytic gene selected from the group consisting of BALF5, BALF2, BMRF1, BSLF1, BBLF4, BBLF 2/3, BcLF1, BZLF1, BRLF1, and BMLF1, or combinations thereof .

5. The human lymphocytes immortalized according to claim 4, characterized in that the recombinant plasmid further comprises EBV genes selected from the group including LMP2a, LMP2b, EBER1, EBER2, EBNA-LP and EBNA3b, or combinations thereof.

6. A method for the production of a desired protein that is free of infectious EBV particles, using immortalized human lymphocytes, the method is characterized in that it comprises: (a) obtaining a recombinant plasmid comprising: a prokaryotic plasmid backbone, attached a DNA segment comprising a group of EBV genes including LMP1, EBNA2, EBNA3a, EBNA3c, and EBNAl, and a group of elements acting in the cis position, of EBV, including TR, oriP, and oriLyt; wherein the recombinant plasmid is free of at least one EBV lytic gene selected from the group consisting of BALF5, BALF2, BMRF1, BSLF1, BBLF4, BBLF 2/3, BcLFl, BZLF1, BRLF1, and BMLF1, or combinations thereof; (b) obtaining cooperating cells; (c) the introduction of the recombinant plasmid into the helper cells; (d) the induction of the lytic phase of the EBV life cycle within the helper cells; (e) collecting a population of viral particles released from the cooperating cells Used; (f) obtaining human lymphocytes; (g) infection of human lymphocytes with the population of viral particles, to form immortalized clones of human lymphocytes; (h) culturing the immortalized clones of human lymphocytes; and (i) the selection of the immortalized clones that produce the desired protein.

7. The method according to claim 6, characterized in the recombinant plasmid further comprises the EBV genes selected from the group including LMP2a, LMP2b, EBER1, EBER2, EBNA-LP and EBNA3b, or combinations thereof.

8. The method according to claim 6, characterized in that a gene encoding a desired protein product is cloned into the recombinant plasmid of step (a).

9. The method according to claim 6, characterized in that the immortalized human lymphocytes are inserted into a human container to produce a desired protein in situ.

10. The method according to claim 8, characterized in that the immortalized human lymphocytes are inserted into a human container, to produce a desired protein in situ. In testimony of which I sign the present in this City of Mexico, D.F., on July 26, 1995. Attorney