METHOD FOR DETECTING REPLICATION COMPETENT VIRUSES IN THE SERA OF SUBJECTS RECEIVING GENE THERAPY

Abstract: The present invention provides a method for detecting replication competent viruses in the sera of subjects receiving gene therapy, the method comprising the step of detecting anti-retroviral antibodies in the sera using a specific antigen. A gene for the antigen exists not in a viral vector for gene therapy but only in a virus genome from which the viral vector originated and the antigen is preferably selected from the group of partial Gag and partial Env proteins of murine leukemia virus. There are also provided antigens whose amino acid sequences include the sequence of SEQ ID NO: 2 or SEQ ID NO: 6, which is of great use in immunoreaction-based RCR detection in view of specificity and sensitivity.

Description: METHOD FOR DETECTING REPLICATION COMPETENT VIRUSES IN THE SERA OF SUBJECTS RECEIVING GENE THERAPY Background of the Invention The present invention relates to a method for detecting replication competent viruses (RCV) during gene therapy by measuring the presence of antibody in the patient serum, using specific antigenic portions of Gag or Env protein of retrovirus, from which viral vectors for gene therapy originated. Gene therapy is a medical intervention used to cure diseases caused by gene mutations or metabolic disorders by introducing normal genes into patient cells. As the biological and genetic engineering technologies will undoubtedly continue to advance rapidly in the future, gene therapy is expected to become the most powerful method for curing diseases which are currently incurable or hard to cure. The most important part of gene therapy is the vector system, which is utilized to introduce therapeutic genes into organisms. Viral vectors including retrovirus, adenovirus and adeno-associated virus are widely used at present. Retroviral vectors are the most frequently employed and are currently used in approximately 50% of clinical trials. Most retroviral vectors have originated from murine leukemia virus (MLV). Though viral vectors are typical choices of gene therapy, certain safety issues surround their use. The therapeutic viral vectors are designed to be replication-defective by genetic engineering, but replication competent virus (RCV) can be generated by homologous recombination between the vector and the patient's nucleotide sequence similar with the viral genome. This is a serious danger against gene therapy using viral vectors. Therefore, in order to ensure the safety of the gene therapy, several methods of analysis have been developed to detect RCV. The US Food and Drug Administration (FDA) guideline requires that the presence of replicating competent retrovirus (RCR) be screened for, not only at the stage of recombinant virus production, but also periodically after administration of viral vectors to patients. The presence of RCR should be screened at two stages. The first stage is at the production step of viral vectors from producer cell lines or before transducing target cells with retroviral vector in vitro. The second stage is after administering genetically engineered cells into subjects wherein the cells contain therapeutic genes introduced by retroviruses. Typically, 10-20 subjects would participate in phase I clinical trial and would be monitored for the presence of RCR three times a year. As such, a great deal of labor, time and materials are depleted during this process. Several methods have been used for RCR detection, including assays using Mus dunni cells, reverse transcriptase assay, and polymerase chain reaction (PCR). The RCR assays using Mus dunni cells are as follows. Virus- producing cells or culture supernatant containing virus are co-cultivated with Mus dunni cells containing marker genes (e.g. Hygromycin, LacZ). The supernatant obtained from this co-culture is used to infect appropriate target cells to monitor the presence of viruses expressing marker genes (Forestell et al, J. Virol. Methods 60:171-178, 1996). This method is based on the principle that RCR containing marker genes can be generated only by recombination between retroviruses that have packaging sequences but not other viral sequence and Mus dunni cells that do not have packaging sequences. Mus dunni cells are also used for S + L " focus assay (Haapala DK et al, J. Virol. 53:827-833, 1985). RCR can be detected by the formation of focus after co-cultivating virus-producing cells or culture supernatant obtained from virus-producing cells with Mus dunni cells. These methods take more than one month because several rounds of co-cultivation of supernatant or virus-producing cells with Mus dunni cells are required to increase the titer of RCR. In addition, S + L " assay itself takes another 5-7 days. The reverse transcriptase assay method depends on the assumption that only RCR generated by recombination can have reverse transcriptase activity, and therapeutic retrovirus cannot. The PCR method detects RCR by performing PCR using a pair of primers, one of which sequences is present in the retroviral vector, while the other is present in the original virus but not in the retroviral vector. This is the most popular method used in clinical trials, but comes with its own problem of low reproducibility (Long et al, Human Gene Ther. 9:1165-1172, 1998; Otto et al, Human Gene Ther. 5:567-575, 1994). All the methods described above require both lengthy time and a great amount of labor, and suffer from the problems of low reproducibility and sensitivity. Therefore, studies have been conducted to develop methods that are more economic, require shorter time and fewer amounts of samples, yet still capable of maintaining a high degree of sensitivity. One such study has reported that RCR can be detected by measuring the antibody generated against MLV using ELISA (Martineau et al, Human Gene Ther. 8:1231- 1241, 1997). However, the specificity and sensitivity of this method are still not satisfactory, because whole MLV virus was used as an antigen. Therefore, the production of highly specific and sensitive antigens is required to solve the problems of specificity, sensitivity and the ensuing restricted usage of antigens in an RCR assay based on antigen-antibody interaction. To overcome the problems of previous RCR detection methods, the inventors have developed a high throughput RCR detection method which can easily screen the generation of antibody after retroviral gene therapy by reacting patient sera with specific antigens derived from MLV. This method is based on the finding that certain fractions of Gag or Env protein have both the specificity and sensitivity levels required for an RCR assay. Summary of the invention Accordingly, it is an objective of the present invention to provide an accurate but simple method for detecting the generation of RCR in a subject. A further object of the present invention is to provide a highly sensitive and economic method that is time efficient and uses a minimum amount of samples in detecting RCR in a subject. A still further object of the present invention is to provide a method that can detect RCR through the reaction of patient sera and antigens derived from Gag or Env of MLV, by measuring the presence of antibody generated from immune reaction against RCR in a subject. Brief description of the drawings Fig. 1 shows the genome structure and gene products of MLV. Fig. 2 represents the schematic diagram of construction of recombinant plasmids in which corresponding DNA fragments encoding viral proteins were inserted into pMAL-c2 vectors so that p30 (CA) of MLV Gag or pl5E (TM) of Env could be expressed as maltose binding protein (MBP) fusion proteins. Fig. 3 illustrates the schematic diagram of construction of recombinant plasmids in which corresponding encoding DNAs were inserted into pET-17b or pET-3a vector so that they were able to express CA, TM or gp70 (SU-1) of MLV Env as T7 tagged proteins. Fig. 4a is a photograph of SDS-PAGE showing the expression level of MLV Gag (CA) protein in E. coli before and after IPTG induction. Lane 1: control (pMAL-c2 induced with ITPG) Lane 2 and 3: before and after IPTG induction of MBP-CA (70 kDa) Lane 4 and 5: before and after IPTG induction of T7-CA (31 kDa) Fig. 4b is a photograph of SDS-PAGE demonstrating the expression level of MLV Env (TM and SU-1) protein in E. coli before and after IPTG induction. Lane 1 : control (pMAL-c2 induced with ITPG) Lane 2 and 3: before and after IPTG induction of MBP-TM (57 kDa) Lane 4 and 5: before and after IPTG induction of T7-TM (17 kDa) Lane 6 and 7: before and after IPTG induction of T7-SU-1 (24 kDa) Fig. 5 is a photograph of gel exhibiting the result of purification through column chromatography (amylose resin column for MBP fusion protein, and Sepharose column for T7-tagged protein) after harvesting antigenic proteins produced in E. coli. Lane 1 : CA in which MBP portion was removed by protease Factor Xa Lane 2: T7-CA Lane 3 : MBP-TM Lane 4: T7-TM Lane 5: T7-SU-1 Fig. 6a is a photograph displaying the result of Western blotting in which recombinant proteins CA, TM and SU-1 expressed from pMAL-c2, pET-17b or pET-3a were reacted with rabbit anti-CA serum. Lane 1: CA Lane 2: T7-CA Lane 3: MBP-TM Lane 4: T7-TM Lane 5: T7-SU-1 Fig. 6b is a photograph showing the result of Western blotting in which recombinant proteins CA, TM and SU-1 expressed from pET-17b or pET-3a were reacted with mouse anti-MLV serum. Lane 1: T7-CA Lane 2: T7-TM Lane 3: T7-SU-1 Fig. 7a is a graph showing the specificity of T7-CA as S/CO (ratio between absorbance of sample and that of cutoff value) after ELISA in which rabbit anti-CA serum served as a positive sample and normal rabbit serum as a negative control. Fig. 7b is a graph representing the specificity of T7-SU-1 as S/CO after ELISA in which mouse anti-MLV serum served as a positive sample and normal mouse serum as a negative control. Fig. 7c is a graph illustrating the specificity of T7-CA and T7-SU-1 after ELISA in which each protein alone or together were used as antigens, and reacted with mouse anti-MLV serum. Fig. 8 is a photograph exhibiting the results of the specificity test in which T7-CA, T7-TM and T7-SU-1 were fabricated in the form of protein chips, and reacted with rabbit anti-CA serum (a), mouse anti-MLV serum (b), and normal mouse serum (c). Detailed description of the invention The present invention provides a method for detecting replication competent viruses (RCV) during gene therapy by measuring the presence of antibody in the patient serum, using some antigenic portions of Gag or Env protein of retrovirus, from which retroviral vectors for gene therapy originated. The inventive RCV detection method measures antibodies present in the patient sera through an antigen-antibody reaction. The gene encoding an antigenic protein that can be used in this reaction to react with patient sera is not present in the viral vector used in gene therapy but present in the viral genome from which the vector originated. Viral vectors used in gene therapy are ordinarily unable to replicate because most of gag, pol and env are removed from the original virus genome. The transfection step into packaging cells, which can supply Gag, Pol, and Env in trans, is required to package them into viral particles. Therefore, portions of Gag or Env are preferred as antigenic proteins because genes encoding them are not present in the viral vector used in gene therapy but present in the viral genome from which the virus vector originated. It is therefore possible to eliminate false positive signals that may otherwise be generated from the viral vector itself, rendering the inventive method highly specific. The RCR detection method of the present invention can be applied to all cases in which any of retrovirus, adenovirus, adeno-associated virus, and lentivirus is used as a therapeutic vector. Also, the inventive RCR detection method can be applied to gene therapy which uses retrovirus, especially murine leukemia virus (MLV). It is particularly preferable that the antigens are portions of the MLV Gag or Env protein. It is more preferable for higher specificity and sensitivity that the portion of MLV Gag protein includes the amino acid sequence represented by SEQ ID NO: 2, and that the portion of MLV Env protein includes the amino acid sequence represented by SEQ ID NO: 6. Above antigenic proteins can be expressed as fusion proteins - fused with maltose binding protein or T7 tag that is comprised of 11 amino acids - in order to facilitate purification. The inventive RCR detection method based on an antigen-antibody reaction may be practiced by way of using various procedures which include ELISA (enzyme-linked immunosorbent assay), Western blotting, FLISA (fluorescence-linked immunosorbent assay), and other analytical methods which can detect antigen-antibody binding. Although ELISA and FLISA are preferably used, Western blotting or neutralization assay coupled with ELISA or FLISA can increase the accuracy of the analysis. Again, the coupling of the present invention with the known PCR method can be used to obtain the same effect. The antigenic proteins of the present invention can be fabricated for analysis in the form of protein chips using an automatic microarrayer. High-throughput, automation, and efficacy of reaction can be achieved when a protein chip is employed because a large number of samples can be analyzed in one reaction using a single chip. The protein chip is a small- scale solid matrix, on which dozens to several hundred spots are arrayed, each spot containing 1 to 100 pg of an antigen and fixed on the small-scale solid matrix. The chip can be utilized for mass-analysis because it can be produced using an automatic microarrayer. The substrate used for the production of the said protein chip can be made of various materials. Preferable materials include, but are not limited to, glass, modified silicones, commonly used polymers (e.g. tetrafiuoroethylene, polystyrene, and polypropylene) and gels. Although the preferred substance to coat the micro-substrate with is aminoalkylsilane, other substances can be also used, including plastics, resins, carbohydrates, silica, silica derivatives, carbon, metals, inorganic glass, membranes or chemicals for the immobilization of antigenic proteins. A protein chip coated with antigens can be immersed in alcohol having a concentration of more than 50%. On performing RCV analysis using said protein chip coated with antigens, mass-analysis could be carried out if the patient sera react with antigens using microarrayer, then with anti- human IgG antibody conjugated with fluorescent dye as a secondary antibody. The result would be obtained using an automatic reader such as a high-speed fluorescence scanner. The present invention also provides a diagnostic kit that can analyze the generation of RCV in patient sera receiving gene therapy. The inventive kit contains antigens, genes encoding them being not present in the viral vector used in gene therapy but present in the viral genome from which the vector originated. As antigenic proteins for the above process, portions of MLV Gag or Env protein are preferable. In particular, a portion of Gag protein including the amino acid sequence represented by SEQ ID NO: 2 or a portion of MLV Env protein including the amino acid sequence represented by SEQ ID NO: 6 is more preferable. The said RCR diagnostic kit can be provided in the form of a protein chip in which antigenic proteins are fixed on a small-scale solid matrix. The said kit can include additional suitable reagents or apparatuses that are generally required for ELISA, FLISA, or Western blotting. The antigenic proteins for RCV detection method of the present invention can be produced on a large scale by constructing expression plasmids which contain some genes from viral genomes utilized in the production of therapeutic recombinant virus, and by expressing those genes in E.coli and so forth. The present invention provides an expression vector containing the nucleotide sequence of SEQ ID NO: 1 (encoding the antigen of SEQ ID NO: 2) and an expression vector containing the nucleotide sequence of SEQ ID NO: 5 (encoding the antigen of SEQ ID NO: 6), which were deposited in the Korean Culture Center of Microorganisms under Deposit Nos: KCCM-10173 (November 11, 1999) and KCCM- 10224 (November 10, 2000). p30 (capsid protein; CA, portion of MLV Gag protein), pl5E (TM), and gp70 (upstream including Pro-rich sequence; SU-1) were selected as antigens to detect anti-MLV antibody in the preferable examples of the present invention. Capsid protein p30 is known to be highly expressed in most retroviruses, and the amino acid sequence is highly conserved among different subtypes. Env antigens were selected as major antigens of diagnostic reagents. Although the amino acid sequence of Env is less conserved than that of the capsid protein, the immunogenicity is expected to be higher. Also, both the transmembrane domain (TM) and the extracellular domain (SU-1) of the Env protein were cloned because the antigenicity of both domains appeared to be different. The positions of selected antigens (CA, TM, and SU-1) in the genomes are illustrated in Fig. 1. Highly conserved regions were selected based on the amino acid sequence of MLV Gag and Env. Then, genes encoding parts of CA, TM, and SU-1 were generated by PCR, and inserted into expression vectors. pMAL-c2 vectors that can make the MBP fusion proteins, pET17b and pET3a expressing T7-tagged proteins, were used as expression vectors for the convenience of protein purification. The results of protein expression in E.coli showed that the expression levels and antigenicity of proteins varied between sequences. Most notably, CA which consisted of 263 amino acids and SU-1 which consisted of 197 amino acids showed highest expression level and antigenicity. On the other hand, the expression level of the SU- 1 protein inserted into pET-17b was too low to be used in further experiment. Expression of protein in E.coli was induced by the addition of IPTG, detected in SDS-PAGE, and the protein was then purified by column chromatography. The expression levels of antigenic proteins in the present invention were very low before the addition of IPTG, but they increased to 10-20% of the total protein after addition of IPTG. The antigens were detected in almost single bands after purification, indicating high level of purity (see Fig. 5). Western blotting and ELISA were performed to check the specificity and sensitivity of the antigens to anti-MLV antibody in other examples of the present invention. Fusion protein MBP-CA was treated with protease Factor Xa to obtain pure CA protein. Purified CA was i.p. injected into rabbits, and rabbit sera containing anti-CA antibody was obtained. Anti- MLV antibody was produced by injecting MLV (as a whole virus) into the tail vein of mice and harvesting positive mouse sera from the immunized mice. Rabbit anti-CA sera and mouse anti-MLV sera were used in the examples, as the cases of MLV infection in humans are extremely rare, and it is impossible to obtain positive human sera. Western blotting was performed by reacting antigenic proteins of the present invention with the said rabbit anti-CA. serum or mouse anti-MLV serum. The results showed that each antigen reacted specifically with the antibody (see Figs. 6a and 6b). In the case of Gag, both CA and T7-CA reacted very strongly with a 1 :5000 dilution of rabbit serum. However, TM and SU-1 of the Env protein did not react. Again, only T7-CA and T7-SU-1 reacted with mouse anti-MLV serum. SU- 1 showed stronger reactivity than CA between the two proteins. T7-TM did not react at all. The sensitivity and specificity of the assay were assessed by ELISA with antigenic proteins and antibodies in other examples of the present invention. The cutoff of ELISA was 0.3 above the mean absorbance of negative controls (normal rabbit sera or normal mouse sera). The results were presented as S/CO (sample OD/cutoff value), and when S/CO was higher than 1 , it was regarded as positive. S/CO of CA and T7-CA to rabbit anti-CA serum was higher than 4, and regarded as positive, but S/CO of MBP-TM, T7-TM, and T7-SU-1 to it were lower than 1, and regarded as negative. These results are consistent with Western blotting in previous experiments and show that the CA antigen of the present invention has high specificity. S/CO of CA, T7-CA, and T7- SU-1 to mouse anti-MLV serum was higher than 3, and S/CO of MBP-TM and T7-TM was between 1 and 2. These results suggested the difference of these proteins in their antigenicity. On the basis of these results, the sensitivity of ELISA was assessed with T7-CA and T7-SU- 1 , which showed high antigenicity. The sensitivity of these antigens was assessed in ELISA by two-fold serial dilutions of specimen. ELISA was performed with rabbit serum which was diluted up to 1 : 640,000, and with mouse serum diluted to 1 :2560. The initial mouse serum was 10-fold diluted in human serum to simulate the situation of human exposed to MLV. S/CO of T7-CA was higher than 3 up to a 1: 160,000 dilution of rabbit anti-CA serum (see Fig. 7a), showing high sensitivity. S/CO of T7-SU-1 was higher than 4 up to a 1 :64 dilution of mouse anti-MLV serum (see Fig. 7b). S/CO of T7-CA and T7-SU-1 was higher than 3 up to a 1 :64 dilution and a 1 :256 dilution of mouse anti-MLV serum, respectively. When two antigens were mixed, S/CO was higher than 2 up to a 1:256 dilution (see Fig. 7c). From the above results, it has been shown that CA and SU-1 antigens have both a high sensitivity and a high specificity, demonstrating that they can be used to identify RCR which can be generated through the homologous recombination by detecting MLV antibody in the sera of patient who have been subjected to gene therapy. The decrease of S/CO of T7-SU-1 with the dilution of sera was slower than that of T7-CA. This suggests that even if T7-SU-1 were to be used as a single antigen, it could function as an effective antigen for detection of any anti-MLV antibodies. In other examples of the present invention, protein chips in which T7- C A, T7-TM, and T7-SU- 1 antigens were fixed onto aminoalkylsilane-coated slide glasses were used to investigate the possibility of application to the mass-analysis. T7-CA and T7-SU-1 showed high sensitivity and specificity in antigen-antibody reaction on protein chips, and it has been confirmed that the antigens of the present invention are suitable for mass-analysis of RCR using protein chips. The present invention is described in detail in the Examples below. It should be evident to people of common knowledge in the area in which the present invention was conducted that the following Examples are intended to further illustrate the present invention without limiting its scope. Examples Example 1: Cloning of MLV antigens and construction of expression vectors DNA fragments encoding capsid protein (CA) p30, pl5E (TM), and gp70 (upstream region of proline-rich sequence; SU-1) of MLV were amplified by PCR before large scale production of antigens and efficiency of these antigens to detect RCR has been confirmed. pMLV (Shinnick et al, Nature 293:543-548, 1981) was used as a template, and synthetic oligonucleotides containing appropriate restriction enzyme sites were used as 5' and 3' primers for amplification of CA, TM, and SU-1. The sequences of primers used in PCR are presented in Table 1 as SEQ ID NOs: 7-14 according to the target genes and restriction enzyme sites. Table 1 PCR was performed using MLV HIT456 genome as a template. Reactions consisted of 30 cycles of a denaturation step at 94°C for 1 min, an annealing step at 55°C for 1 min, and an extension step at 72°C for 1 min 30 sec. The reaction contained 1 μl of each primer (10 pmol/μl) and 0.75 mM High Fidelity PCR system (Roche, USA). PCR products were ligated into pCR Blunt (Invitrogen, The Netherlands) or pCR2.1 (Invitrogen, The Netherlands). To describe in detail, CA gene fragment amplified by a combination of primers of SEQ ID NOs: 7 and 9, or 7 and 8, and TM gene fragment amplified by a combination of primers of SEQ ID NOs: 10 and 12, or 11 and 12 were inserted into pCR Blunt vectors. As well, SU-1 gene fragment amplified by a combination of primers of SEQ ID NOs: 13 and 14 was inserted into a pCR2.1 vector. Genes described above were cloned into pMAL-c2 (New England Biolabs, USA), pET-17b (Novagen, USA), or pET-3a (Novagen, USA) in order to express antigens in the form of MBP (maltose binding protein) fusion proteins, or T7-tagged (Met-Ala-Ser-Met-Thr-Gly-Gly-Glu-Glu-Met- Gly) proteins. The construction schemes of the expression vectors are shown in Figs. 2 and 3. Firstly, CA PCR product (768 bp), amplified by a pair of primers of SEQ ID NOs: 7 and 9 was inserted into the EcoRI/Sall site of pMAL-c2, resulting in MBP-CA. CA PCR product (734 bp) amplified by a pair of primers of SEQ ID Nos: 8 and 9 was inserted into the BamHI/XhoI site of pET- 17b to produce T7-CA. TM PCR product (396 bp) amplified by a pair of primers of SEQ ID NOs: 10 and 12 was inserted into the EcoRI/Sall site of pMAL-c2, generating MBP-TM. TM PCR product (369 bp) amplified by a pair of primers of SEQ ID NOs: 11 and 12 was inserted into the BamHI/XhoI site of pET-17b to produce T7-TM. SU-1 PCR product (591 bp) amplified by a pair of primers of SEQ ID NOs: 13 and 14 was inserted into the BamHI site of pET-3a, resulting in T7- SU-1. Expression plasmids derived from pET-17b and pET-3a were transformed into the E.coli BL21 (DE3) strain. E.coli strain transformed with T7-CA (BL21-T7-p30) and E.coli strain transformed with T7-TM (BL21-T7-pl5E) were deposited in the Korean Culture Center of Microorganisms (Deposit No: KCCM-10173, KCCM-10174) on the date of November 11, 1999. E.coli strain transformed with T7-SU-1 (BL21-T7-gp70) was deposited in the same center (Deposit No: KCCM-10224) on the date of November 10, 2000. Example 2: Expression and purification of MLV antigens in E. coli E.coli transformed with MVL Gag or Env expression plasmids prepared in Example 1 was grown at 37°C in 1 i of LA broth (2% Bactotriptone, 1% yeast extract, 2% NaCl, lx ampicillin) until OD 60 o reached 0.5. Isopropyl-β-D-thiogalactopyranoid (IPTG) was added to a final concentration of 1 mM to induce expression of CA of Gag, TM and SU-1 of Env. Cells were harvested by centrifugation at 5000 rpm for 10 min at 4 °C. Then, the cell pellets were resuspended in lysis buffer (20 mM Tris, 2 mM EDTA, 1 mM phenyl-methylsulfonyl fluoride, pH 8.0), and were placed on ice for 20 min. The cells were lysed by sonication for 5 min, and the supernatant and the precipitate were obtained by centrifugation. SDS-PAGE was performed to identify whether target antigen was present in the supernatant or in the precipitate. The antigen in the supernatant was purified without further steps, but the antigen in the precipitate was purified by two purification methods according to the expression vector. In the case of antigen expressed from pET-17b, the precipitates were solubilized by 8 M urea solution, adjusted to pH 5.6, and loaded into Q Sepharose Fastflow column (Amersham Pharmacia, Sweden) equilibrated with equilibration buffer (8 M urea in sodium acetate, pH 5.6). After loading, the column was washed by more than 3 bed volumes of equilibration buffer. The target antigen was purified by NaCl gradient from 50 mM to 500 mM after monitoring optical density of the eluents. SU-1 antigen expressed from pET-3a was purified using the same scheme except that: the pH of the solution was adjusted to pH 9.6, carbonate buffer containing 8 M urea (8 M urea in sodium carbonate, pH 9.6) was used as an equilibration buffer, and SP Sepharose Fastflow column (Amersham Pharmacia, Sweden) was used in purification. In the case of antigens expressed from the pMAL-c2 vector, the precipitates were solubilized in the amylose resin equilibration buffer (20 mM Tris-HCl, 2 M NaCl, 1 mM EDTA, 10 mM β-mercaptoethanol, pH 7.4), and applied into amylose column (New England Biolabs, USA) equilibrated with equilibration buffer. After loading, the column was washed by more than 10 bed volumes of equilibration buffer. The target antigen was purified by elution buffer (10 mM maltose in equilibration buffer), and optical density of the eluents was measured at 280 nm. The MBP tag was removed by Factor Xa, and the product was used in the later experiment. The purity of antigens obtained through the above processes was confirmed by SDS-PAGE. Very small amounts of antigens were expressed before the addition of IPTG, but the expression level was increased to 10- 20% of the total protein after addition of IPTG (see Figs. 4a and 4b). The antigens were detected almost as single bands following the purification of collected inclusion body using column chromatography to show high purity (see Fig. 5). The expression level of each antigen protein is presented in Table 2. Table 2 SU-1 fragment (591 bp) amplified by a pair of primers of SEQ ID NOs: 13 and 14 which was inserted into pET-17b was not used in the later experiment because the expression level was too low as shown in the above table. Example 3 : Immunization of animals with MLV antigens Viral supernatant obtained from cells transfected with MLV vector and CA antigen derived from MBP-CA by removing MBP tag were used as immunogens in the immunization of animals. Human embryonic kidney cell line (293T cells) was transfected with DO-A2-CAT plasmid by the 3 plasmids transfection method (Yuco et al, Nucleic Acids Res., 23-24, 1995), and supernatant containing virus was prepared by filtrating the media with 0.45 μm membrane filter 48 h after transfection. The tail veins of 20 mice (5 weeks old) were injected with 100 μl of viral supernatant 4 times for 2 weeks. For CA antigen, CA part was obtained by treatment with Factor Xa of MBP-CA which was purified in Example 3. It was mixed with Freund's complete adjuvant (GibcoBRL, USA) 1 :1 ratio, and was i.p. injected into 2 rabbits (8 weeks old) at the concentration of 100 μg/ml 3 times for 4 weeks. Blood was drawn from the hearts of the animals after immunization in order to obtain sera against MLV, and the sera were used in Western blotting and ELISA together with the purified antigens. Example 4: Western blotting Antigen-antibody interaction was analyzed by Western blotting using CA, TM, and SU-1 antigens and antibodies to CA or MLV obtained in Example 3 to test if MLV antigens produced in Example 2 could react effectively with MLV antibodies. Rabbit anti-CA serum and mouse anti- MLV serums were used in the experiment because the incidence of MLV infection in humans is very rare. Purified antigens were resolved by SDS-PAGE with minigel apparatus (#SE280, Hoefer Pharmacia Biotech, USA), and transferred to a nitrocellulose membrane. The membrane was incubated in a blocking buffer (5% skim milk, 0.1 % Tween 20, Tris buffered saline) for 1 h with slow shaking, and was incubated for 1 h at room temperature with a 1 :5000 dilution of rabbit anti-CA serum or a 1 :500 dilution of mouse anti-MLV serum. The membrane was washed 3 times with TBST and incubated for 30 min with a 1 : 10000 dilution (in blocking buffer) of horseradish peroxidase (HRP) conjugated goat anti-rabbit or anti-mouse IgG antibody. Next, the membrane was washed 3 times with TBST. 1 ml of ECL solution (Luminol/Enhancer and stable peroxide solution, Amersham) was then added to it. It was then incubated 1 min at room temperature, exposed to film in a dark room, and the bands were visualized using developing solution. From the result of Western blotting, the purified antigens showed specific interaction with the antibodies. In the case of Gag, both CA and T7-CA interacted strongly with a 1 :5000 dilution of rabbit serum. Neither TM nor SU-1 interacted with the rabbit serum (see Fig. 6a). Both T7-CA and T7-SU-1 showed interaction with mouse anti-MLV serum. SU-1 showed stronger reactivity than CA. T7-TM failed to show any interaction (see Fig. 6b). Example 5: ELISA The diagnosis of viral infection is generally performed by ELISA. ELISAs were carried out to determine whether the antigens of the present invention have the specificity and sensitivity required in the ELISA of RCR detection. The antigens purified in Example 2 were diluted to a final concentration of 5 μg/ml in 50 mM sodium carbonate buffer (pH 9.6), then 100 μl of diluted antigen was added to each well of 96 well microplate, and placed for 18 h at room temperature. Then 100 μl of phosphate buffered saline (PBS) (pH 7.2) containing 0.5% bovine serum albumin (BSA) was added to each well. After 1 h incubation at room temperature, serum samples were added to the plate. Rabbit anti-CA serum and mouse anti- MLV serum served as positive controls, and normal rabbit or mouse serum served as negative controls. 100 μl of a 1 :1000 and a 1 : 10 dilution of each serum (0.25% Tween 20 in blocking buffer) was added into each well, and incubated for 1 h in a 37°C incubator. After incubation, the plates were washed 5 times with PBST (0.05% Tween 20 in PBS), and 100 μl of a 1 : 10000 dilution of HRP-conjugated goat anti-rabbit or anti-mouse IgG antibody was added to each well. The plate was incubated for 30 min in a 37°C incubator, and washed 5 times with PBST (0.05% Tween 20 in PBS). Then 100 μl of tetramethoxybensidine (TMB) solution was added, and incubated for 30 min at room temperature. The reaction was stopped by addition of 100 μl of 0.5 N H 2 S0 . Absorbance was measured at 450 nm. The cutoff value of ELISA in this experiment is 0.3 above the mean value of the negative control. The result was given as S/CO (sample OD/cutoff value), and the sample was regarded as positive when S/CO was greater than 1 (Table 3). Table 3 S/CO of CA and T7-CA to rabbit anti-CA serum was higher than 4, and regarded as positive. However, S/CO of MBP-TM, T7-TM, and T7- SU-1 was lower than 1, and regarded as negative. The results agree with those of Western blotting, and show that CA antigen used in the present invention has very high specificity. The antigenicity of proteins used in the experiment was different because S/CO of CA, T7-CA and T7-SU-1 to mouse anti-MLV serum was higher than 3, but S/CO of MBP-TM and T7- TM was between 1 and 2. On the basis of the results, the sensitivity of ELISA was assessed using T7-CA and T7-SU-1, which showed high antigenicity. Example 6: Sensitivity of ELISA of CA and SU-1 From the result in Example 5, T7-CA and T7-SU-1 showed very high specificity to rabbit anti-CA and mouse anti-MLV serum. The sensitivity of antigen was assessed by ELISA because it is a very important factor in mass analysis. Two-fold serial dilutions of serum samples were added to the plates coated with T7-CA and T7-SU-1 which showed good reactivity in previous experiments, and ELISA was performed as described in Example 5. Rabbit serum was diluted to a final dilution of 1 : 640000, and mouse serum to a final dilution of 1 :2560. The initial mouse serum was 10-fold diluted in human serum to simulate the situation of human exposed to MLV. The sensitivity of antigens were described as S/CO value in Fig. 7; S/CO of T7-CA to rabbit anti-CA is given in Fig. 7a, S/CO of T7-SU-1 to mouse anti-MLV in Fig. 7b, and S/CO of T7-CA and T7-SU-1 to mouse anti- MLV in Fig. 7c, respectively. T7-CA showed high sensitivity; S/CO was higher than 3 up to a 1:640000 dilution of rabbit anti-CA serum, while S/CO was lower than 1 with normal rabbit serum as shown in the figure (Fig. 7a). T7-SU-1 was negative to normal mouse serum, but positive to mouse anti-MLV serum, and S/CO was higher than 4 up to a 1 :64 dilution (Fig. 7b). S/CO of T7-CA and T7-SU-1 was higher than 3 up to a 1 :64 dilution and a 1 :256 dilution of mouse anti-MLV serum, respectively. When two antigens were mixed, S/CO was higher than 2 up to a 1 :256 dilution (Fig. 7c). These results demonstrate that CA and SU-1 antigens have enough levels of sensitivity and specificity to be used to identify RCR which can be generated through homologous recombination by detecting MLV antibody in patient sera receiving gene therapy. S/CO of T7-SU-1 was decreased with dilutions at a slower rate than that of T7-CA. Therefore, even if T7-SU- 1 were to be used as a single antigen, it could function as an effective antigen for the detection of anti- MLV antibody. Example 7: FLISA using a protein chip Numerous samples could be analyzed in a short period of time if protein chips produced by automatic microarrayers would be used in RCR detection using the antigens of the present invention. To investigate the possibility of using such protein chip for the purpose of RCR detection, the inventors manufactured protein chips by fixing the purified antigens on slide glasses using the GMS 417 Arrayer™ (Genetic MicroSystems, USA) and assessed the reactivity by FLISA. T7-CA, T7-TM, and T7-SU-1 were added to 96 well plates in the concentration of 100 μg/ml, and the plates were set in a microarrayer. 10 pi of each antigen was spotted onto a 2.5 x 7.5 cm microscope slide glass (Sigma, USA) containing 4 sets consisting of 400 spots per set. Spotting was performed in an array mode using 4 pins and rings, and slide glasses coated with aminoalkylsilane were used. The slide glass containing spots were placed at room temperature for 40 min, and then the coated antigens were fixed by immersing the slide glass in 100 % ethanol. The antigens were fixed by bonding amino groups of antigens to alkyl groups of aminoalkylsilane coated on the slide glasses through ethanol treatment, and the slide glasses were dried completely. ELISA was performed using the resulting protein chips for each antigen as described in Example 5. The rabbit anti-CA serum, mouse anti- MLV serum and normal mouse serum were employed in the experiment. Single antibodies were reacted for each chip, and the antibodies spotted on a parafilm were adhered closely to a surface of the slide glass in order to apply the antibodies uniformly. After the antigen-antibody reaction, the fluorescence was detected using a high-speed fluorescence scanner GMS 418 Array Scanner (Genetic MicroSystems, GM200), that was produced from fluorescence-conjugated anti-rabbit or anti-mouse IgG antibody. The surface of the slide glass was read at 532 nm with a flying objective microscope and the obtained data were analyzed using Imagene™ (Genetic MicroSystems, version 2.0), software for analyzing the expressed data (see Fig. 8). Fig. 8a shows the result using rabbit anti-CA serum, while Figs. 8b and c give the result using mouse anti-MLV serum and normal serum. T7- CA reacted strongly with both rabbit anti-CA serum and mouse anti-MLV serum. On the other hand, T7-TM did not react with rabbit anti-CA serum, and reacted with mouse anti-MLV serum. However, the reactivity of T7- TM with mouse serum was lower than that of T7-CA. T7-SU-1 showed a negative reaction with rabbit anti-CA serum, and a strong positive reaction with mouse anti-MLV serum. These results are consistent with the ELISA results in Examples 5 and 6. T7-CA, T7-TM, and T7-SU-1 did not react with normal mouse serum. From the above results, it has been clearly shown that CA and SU-1 antigens of the present invention have both high sensitivity and specificity. BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE INTERNATIONAL FORM r To, Suπyouπg Kim Yongsangu, Echoπdong 300-127 Haπgahg Mansion AFT 18-302 RECEIPT IN THE CASE OF AN ORIGINAL Seoul, issued pursuant to Rule 7, 1 by the Republic of Korea INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page date on which the microorganism was received by the international depositary authouity. BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE INTERNATIONAL FORM To. Sunyσung Kim YotLgsaagu, Echondong 300-127 HaftgaHg 1 Mansion APT 18-302 RECEIPT IN THE CASE OF AN ORIGINAL Seoul, issued pursuant to Rule 7, 1 by the Republic of Korea INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page date on whic t e microorganism was received fay the international depositary authouhy. BUDAPEST TREATY ON THE INTEKNATIONAL RECOGNΓΠON OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE INTERNATIONAL FORM To. Sttnyoung Kim RECEIPT IN THE CASE OF AN ORIGINAL Yongsang Ichoπdong issued pursuant to Rule 7. 1 by the Hangang Mansion AFT 18-302 INTERNATIONAL DEPOSITARY AUTHORITY Seoul, Korea Identified at the bottom of ώis page 1 Where RUle 6. (d) applies, such date is the date on which the status of international depositary authority was acquired '• where a deposit made outside the Budapest Treaty after the acquisition of the status of i nternatioiial depositary authority is converted into a deposit under the Budapest Treaty, such date is the date on which the microorganism was received by the international depositary authouity.

Claims: What is claimed is: 1. A method for detecting a replication competent virus in a subject receiving gene therapy using a viral vector, which comprises reacting the subject's serum with an antigen protein encoded by a gene that is not present in the viral vector but present in the genome of the virus from which the viral vector is derived; and measuring the antibody protein in the subject's serum. 2. The method of claim 1, wherein the viral vector is derived from a retrovirus, an adeno virus, an adeno-associated virus or a lentivirus. 3. The method of claim 2, wherein the retrovirus is murin leukemia virus. 4. The method of claim 1, wherein the antigen protein is a portion of Gag or Env protein of murine leukemia virus. 5. The method of claim 4, wherein the portion of Gag protein of murine leukemia virus has the amino acid sequence of SEQ ID NO: 2. 6. The method of claim 4, wherein the portion of Env protein of murine leukemia virus has the amino acid sequence of SEQ ID NO: 6. 7. The method of claim 1, wherein the antigen protein is in the fusion protein form with a maltose binding protein or a T7 tag consiting of 11 amino acid residues. 8. The method of claim 1, wherein the antigen-measuring step is conducted by a procedure selected from the group consisting of enzyme- linked immunosorbent assay, Western blotting and fluorescence-linked immunosorbent assay. 9. The method of claim 1, which further comprises fixing the antigen to a protein chip using an automated microarrayer before the reacting step. 10. A diagnostic kit for detecting a replication competent virus in a subject receiving gene therapy using a viral vector, which comprises an antigen protein encoded by a gene that is not present in the viral vector but present in the genome of the virus from which the viral vector is derived. 11. The diagnostic kit of claim 10, wherein the viral vector is derived from a retrovirus, an adenovirus, an adeno-associated virus or a lentivirus. 12. The diagnostic kit of claim 10, wherein the antigen protein is a portion of Gag or Env protein of murine leukemia virus. 13. The diagnostic kit of claim 12, wherein the portion of Gag protein of murine leukemia virus has the amino acid sequence of SEQ ID NO: 2. 14. The diagnostic kit of claim 12, wherein the portion of Env protein of murine leukemia virus has the amino acid sequence of SEQ ID NO: 6. 15. The diagnostic kit of claim 10, wherein the antigen protein is fixed on the micro-substrate of a protein chip. 16. An expression vector comprising the nucleotide sequence of SEQ ID NO: 1 which encodes an antigen protein for detecting a replication competent virus(KCCM-10173). 17. An expression vector comprising the nucleotide sequence of SEQ ID NO: 5 which encodes an antigen protein for detecting a replication competent virus(KCM- 10224).

Inventors: KIM SUN-YOUNG, NA YOUNG-SOON, PARK EUN-JIN, YU SEUNG-SHIN, KIM, SUN-YOUNG, NA, YOUNG-SOON, PARK, EUN-JIN, YU, SEUNG-SHIN
Applicants: KIM SUN YOUNG, NA YOUNG SOON, PARK EUN JIN, YU SEUNG SHIN, KIM, SUN-YOUNG, NA, YOUNG-SOON, PARK, EUN-JIN, YU, SEUNG-SHIN
Application year: 2000
Main classification: C12Q1
IPC codes: C07K16, C12N15, C12Q1, G01N33