Abstract:
Tolerance of the immune system for endogenous CD20 can be overcome and an immune response stimulated by administration of xenogeneic or xenoexpressed CD20 antigen. For example, mouse CD20, or antigenically-effective portions thereof, can be used to stimulate an immune response to the corresponding differentiation antigen in a human subject. Administration of xenogeneic antigens in accordance with the invention results in an effective immunity against CD20 expressed by the cancer in the treated individual, thus providing a therapeutic approach to the treatment of lymphomas and leukemia expressing CD20.

Description:
[0001]    This application is a continuation-in-part of U.S. patent application Ser. No. 09/627,694, filed Jul. 28, 2000, which is continuation-in-part of U.S. patent application Ser. No. 09/308,697, filed May 21, 1999, which is a §371 National Phase of International Application No. PCT/US97/22669 filed Dec. 10, 1997. The application also claims benefit under 35 USC §119(e) of U.S. Provisional Application No. 60/036,419 filed Feb. 18, 1997. All of the aforementioned applications are incorporated herein reference. 
     
    
     
       FIELD OF THE INVENTION  
         [0002]    This application relates to a method and compositions for stimulation of an immune response to CD20.  
         BACKGROUND OF THE INVENTION  
         [0003]    Most tumor immunity is mediated by recognition of self-antigens, antigens present in cancer cells that are also found in normal host tissue. Houghton, A. N.,  J. Exp. Med.  180: 1-4 (1994). This type of immunity is more akin to autoimmunity than to immunity in infectious diseases, where the response is directed at a truly foreign antigen, present in the pathogen but not in host tissue. Evidence of this can be found in the autoimmune sequelae that often follow the development of successful tumor immunity. Bowne, W. B., et al.,  J. Exp. Med.  190(11):1717-1722 (1999).  
           [0004]    Differentiation antigens form one prototype of self-antigens in cancer immunity. Houghton, A. N., et al.,  J. Exp. Med.  156(6):1755-1766 (1982). Differentiation antigens are tissue-specific antigens that are shared by autologous and some allogeneic tumors of similar derivation, and on normal tissue counterparts at the same stage of differentiation. Differentiation antigens have been shown to be expressed by a variety of tumor types, including melanoma, leukemia, lymphomas, colorectal, carcinoma, breast carcinoma, prostate carcinoma, ovarian carcinoma, pancreas carcinomas, and lung cancers. Typically the expression of these antigens changes as a cell matures and can characterize tumors as more or less differentiated. For example, differentiation antigens expressed by melanoma cells include Melan-A/MART-1, Pmell7, tyrosinase, gp75 and gplOO. Differentiation antigens expressed by lymphomas and leukemia include CD19 and CD20/CD20 B Iymphocyte differentiation markers. An example of a differentiation antigen expressed by colorectal carcinoma, breast carcinoma, pancreas carcinoma, prostate carcinoma, ovarian carcinoma, and lung carcinoma is the mucin polypeptide muc-1. A differentiation antigen expressed by breast carcinoma is her2/neu. The her2/neu differentiation antigen is also expressed by ovarian carcinoma. Differentiation antigens expressed by prostate carcinoma include prostate specific antigen, prostatic acid phosphatase, and prostate specific membrane antigen (PSMA).  
           [0005]    Unfortunately, in most cases, the immune system of the individual is tolerant of these antigens, and fails to mount an effective immune response. For the treatment of cancers where the tumor expresses differentiation antigens therefore, it would be desirable to have a method for stimulating an immune response against the differentiation antigen in vivo. It is an object of the present invention to provide such a method.  
         SUMMARY OF THE INVENTION  
         [0006]    It has now been found that the tolerance of the immune system for endogenous CD20 can be overcome and an immune response stimulated by administration of xenogeneic CD20 and CD20 (including syngeneic CD20) expressed in cells of different species. For example, mouse CD20, or antigenically effective portions thereof, can be used to stimulate an immune response to the corresponding differentiation antigen in a human subject. Administration of xenogeneic or xenoexpressed antigens in accordance with the invention results in an effective immunity against CD20 expressed by the cancer in the treated individual, thus providing a therapeutic approach to the treatment of lymphomas and leukemia expressing CD20.  
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0007]    [0007]FIGS. 1 a - d  show the maps of four plasmids containing regions encoding a CD20 antigen.  
         [0008]    [0008]FIGS. 2 a - d  show Kaplan-Meier survival curves of studies on CD20 genetic immunization.  
         [0009]    [0009]FIG. 3 shows T cell responses against CD20 in mice immunized with CD20. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0010]    The present invention provides a method for stimulating an immune response to a tissue expressing CD20 in a subject individual. The subject individual is preferably human, although the invention can be applied in veterinary applications to animal species, preferably mammalian (for example horse, dog or cat) or avian species, as well.  
         [0011]    As used in the specification and claims of this application, the term “immune response” encompasses both cellular and humoral immune responses. Preferably, the immune response is sufficient to provide immunoprotection against growth of tumors expressing CD20. The term “stimulate” refers to the initial stimulation of a new immune response or to the enhancement of a pre-existing immune response.  
         [0012]    In accordance with the invention, a subject individual is treated to stimulate an immune response to endogenous CD20 by administering a xenogeneic or xenoexpressed CD20 antigen. The term “xenogeneic” denotes the fact that the administered antigen has a sequence peptide different from the CD20 of the species being treated and originates from a different species. For treatments of humans, preferred xenogeneic antigens will be rodent antigens, for example mouse, but could come from other mammals such as dog, cat, cow, or sheep, or from birds, fish, amphibian, reptile, insect or other more distantly related species. The term “xenoexpressed” refers to an antigen which may be syngeneic with the subject individual, but which is expressed in cells of a species different from the subject individual, for example in insect cells.  
         [0013]    The term “CD20 antigen” refers to a protein/peptide antigen or to a polynucleotide having a sequence that is expressed in vivo to produce the protein/peptide antigen. In either case, the protein/peptide antigen may be the entire CD20 molecule, or some antigenic portion thereof derived from the extracellular domain. For example, as described below, plasmids were prepared using either full length cDNA or using a portion encoding a 43 amino acid extracellular loop (amino acids 136-179 in the mouse protein; amino acids 142-185 in the human protein).  
         [0014]    Administration of a protein/peptide xenogeneic or xenoexpressed CD20 antigen can be accomplished by several routes. First, the xenogeneic CD20 may be administered as part of a vaccine composition which may include one or more adjuvants such as alum, QS21, TITERMAX or its derivatives, incomplete or complete Freund&#39;s and related adjuvants, and cytokines such as granulocyte-macrophage colony stimulating factor (GM-CSF), flt-3 ligand, interleukin-2, interleukin-4 and interleukin-12 for increasing the intensity of the immune response. The vaccine composition may be in the form of xenogeneic CD20 antigen in a solution or a suspension, or the therapeutic differentiation antigen may be introduced in a lipid carrier such as a liposome. Such compositions will generally be administered by subcutaneous, intradermal or intramuscular route. Vaccine compositions containing protein/peptide xenogeneic or xenoexpressed CD20 antigen are administered in amounts which are effective to stimulate an immune response to the target differentiation antigen in the subject individual. The preferred amount to be administered will depend on the species of the target individual and on the specific antigen, but can be determined through routine preliminary tests in which increasing doses are given and the extent of antibody formation or T cell response is measured by enzyme-linked immunosorbent assay (ELISA) or similar tests. T cell responses may also be measured by cellular immune assays, such as cytokine release assays and proliferation assays.  
         [0015]    Xenogeneic CD20 antigen may also be introduced in accordance with the invention using a DNA immunization technique in which DNA encoding the antigen is introduced into the subject such that the antigen is expressed by the subject. Xenogeneic CD20 antigen may also be administered as a purified protein. Proteins can be purified for this purpose from cell lysates using column chromatography procedures. Proteins for this purpose may also be purified from recombinant sources, such as bacterial or yeast clones or mammalian or insect cell lines expressing the desired product.  
         [0016]    Xenogeneic CD20 antigen may also be administered indirectly through genetic immunization of the subject with DNA encoding the antigen. cDNA encoding the xenogeneic CD20 antigen is combined with a promoter which is effective for expression of the cDNA in mammalian cells. This can be accomplished by digesting the nucleic acid polymer with a restriction endonuclease and cloning into a plasmid containing a promoter such as the SV40 promoter, the cytomegalovirus (CMV) promoter or the Rous sarcoma virus (RSV) promoter. The resulting construct is then used as a vaccine for genetic immunization. The cDNA can also be cloned into plasmid and viral vectors that are known to transduce mammalian cells. These vectors include retroviral vectors, adenovirus vectors, vaccinia virus vectors, pox virus vectors and adenovirus-associated vectors.  
         [0017]    [0017]FIGS. 1 a - d  show maps of four plasmids containing regions encoding a CD20 antigen: pCR3-mCD20, pCR3-hCD20, pNERIS-M8, and pNERIS-H8. The preparation and use of these plasmids is described in the examples below.  
         [0018]    The nucleic acid constructs containing the promoter, CD20 antigen-coding region and intracellular sorting region can be administered directly or they can be packaged in liposomes or coated onto colloidal gold particles prior to administration. Techniques for packaging DNA vaccines into liposomes are known in the art, for example from Murray, ed. “Gene Transfer and Expression Protocols” Humana Pres, Clifton, N.J. (1991). Similarly, techniques for coating naked DNA onto gold particles are taught in Yang, “Gene transfer into mammalian somatic cells in vivo”,  Crit. Rev. Biotech.  12: 335-356 (1992), and techniques for expression of proteins using viral vectors are found in Adolph, K. ed. “Viral Genome Methods” CRC Press, Florida (1996).  
         [0019]    For genetic immunization, the vaccine compositions are preferably administered intradermally, subcutaneously or intramuscularly by injection or by gas driven particle bombardment, and are delivered in an amount effective to stimulate an immune response in the host organism. The compositions may also be administered ex vivo to blood or bone marrow-derived cells (which include APCs) using liposomal transfection, particle bombardment or viral infection (including co-cultivation techniques). The treated cells are then reintroduced back into the subject to be immunized. While it will be understood that the amount of material needed will depend on the immunogenicity of each individual construct and cannot be predicted a priori, the process of determining the appropriate dosage for any given construct is straightforward. Specifically, a series of dosages of increasing size, starting at about 0.1 μg is administered and the resulting immune response is observed, for example by measuring antibody titer using an ELISA assay, detecting CTL response using a chromium release assay or detecting TH (helper T cell) response using a cytokine release assay.  
         [0020]    In the experiments described below, the BALB/c mouse lymphoma model, in which the mouse lymphoma cell line A20 is used to challenge the animals after immunization with CD20, was used to test the efficacy of treatment. The intravenous injection of 5×10 4  A20 cells in non-immunized mice results in the development in 3 to 4 weeks of ascites and large palpable tumor masses in the abdomen. This is usually accompanied by weight gain. A few mice develop hind-legs paralysis and wasting. In our studies, groups of 10-12 mice received five weekly immunizations with either the vector alone (pCR3) or one of the following: pCR3-mCD20, pCR3-hCD20, pNERIS-M8 or pNERIS-H8. The latter two plasmids encode for a 43 aa extracellular loop (aa 136-179 in the mouse protein and 142-185 in the human protein). The vaccinations were tolerated without any appreciable side effects other than minimal inflammation of the skin at the site of inoculation. Five days following the last immunization, the mice were challenged with 5×10 4  A20 cells. The first signs of tumor development were observed as expected around the third week after tumor challenge. Results are plotted as Kaplan-Meier survival curves in FIGS. 2 a - d . The groups were compared by log rank test. Mice vaccinated with the vector control plasmid (pCR3) and the full-length mouse cDNA (pCR3-mCD20) developed ascites and tumor masses at a similar pace, and had to be sacrificed between 34 and 45 days post-challenge in three independent experiments. Mice immunized with the human minigene (pNERIS-H8) displayed a slower pace of tumor take, as shown in panel  3   a  and  3   b . Long-term survival, defined as survival up to 60 days post-challenge, was modestly but statistically better in mice immunized with pCR3-H8 ( 3   c  and  3   d , p&lt;0.05 in both cases). Results in mice immunized with the full-length human cDNA (pCR3-hCD20) or the mouse minigene (pNERIS-M8) were more variable, and not statistically significant. These results were consistently reproduced three times.  
         [0021]    T-cell responses against mouse CD20 were observed following vaccination with human CD20 DNA. BALB/c-3T3 fibroblasts were stably transfected with the plasmids expressing full-length human and mouse CD20 (3T3H and 3T3M, respectively) and human and mouse minigenes (3T3HM and 3T3MM), as described. CD8+ T-cells were isolated by positive selection from mice immunized weekly three times with the same constructs. The T-cells were tested for reactivity against each antigen by elispot assay as described in the examples. As shown in FIG. 3, T-cells from mice immunized with pCR3 only did not react against any of the transfectants. Mice immunized with both the full-length mouse CD20 and the mouse minigene had also a background reactivity. The CD8+ T-cells of mice immunized with the human full-length CD20 reacted strongly against not only 3T3H but also with 3T3M, 3T3HM and 3T3MM, as well as against A20, a CD20-expressing cell line. Mice immunized with the human minigene had intermediate responses, with significant reactivity against 3T3H and 3T3MM.  
         [0022]    Antibody responses against mouse CD20 were observed following vaccination with human CD20 DNA. The sera of the immunized mice was collected and analyzed for the presence of anti-mouse CD20 antibodies, a sign of autoimmunity. The sera were immuno-precipitated with recombinant mCD20 protein in the presence of anti-mouse IgG bound to agarose beads. Negative controls for non-specific binding of the anti-mouse IgG did not include recombinant CD20 in the reaction. The precipitated proteins, separated in a 4-20% SDS-PAGE were then blotted with the monoclonal antibody anti-Xpress. The expected size of the recombinant mCD20 is 40Kda. In the sera from mice immunized with syngeneic DNA we could not detect any anti-CD20 antibodies, while four out of five mice immunized with xenogeneic DNA display the presence of auto-reactive anti CD20 antibodies. Analysis of the sera from mice immunized with both human and mouse minigenes revealed the presence of anti CD20 antibodies in about 80% of the animals tested.  
         [0023]    In accordance with a further aspect of the present invention, an immune response against a target differentiation antigen can be stimulated by the administration of syngeneic differentiation antigen expressed in cells of a different species, i.e. by xeonexpressed CD20 antigen. In general, the subject being treated will be a human or other mammal. Thus, insect cells are a preferred type of cells for expression of the syngeneic differentiation antigen. Suitable insect cell lines includes Sf9 cells and Schneider 2 Drosophila cells. The therapeutic differentiation antigen could also be expressed in bacteria, yeast or mammalian cell lines such as COS or Chinese hamster ovary cells. Host cells which are evolutionarily remote from the subject being treated, e.g. insects, yeast or bacteria for a mammalian subject, may be preferred since they are less likely to process the expressed protein in a manner identical to the subject.  
         [0024]    To provide for expression of the differentiation antigen in the chosen system, DNA encoding the differentiation antigen or a portion thereof sufficient to provide an immunologically effective expression product is inserted into a suitable expression vector. There are many vector systems known which provide for expression of incorporated genetic material in a host cell, including baculovirus vectors for use with insect cells, bacterial and yeast expression vectors, and plasmid vectors (such as psvk3) for use with mammalian cells. The use of these systems is well known in the art. For treatment of humans with a syngeneic differentiation antigen, cDNA encoding the human differentiation antigen to be targeted must be available. cDNA is produced by reverse transcription of mRNA, and the specific cDNA encoding the target differentiation antigen can be identified from a human cDNA library using probes derived from the protein sequence of the differentiation antigen. The cDNA sequences human CD20 is known, and is reflected in the sequences provided herein. Xenoexpressed CD20 antigen, like purified xenogeneic CD20 antigen, is administered to the subject individual in an amount effective to induce an immune response. The composition administered may be a lysate of cells expressing the xenoexpressed antigen, or it may be a purified or partially purified preparation of the xenoexpressed antigen.  
         [0025]    The invention will now be further described with reference to the following, non-limiting examples, in which the following material were used:  
         [0026]    Mice: BALB/c mice were purchased from the National Cancer Institute breeding program (Frederick, Md.) or from Taconic (Germantown, N.Y.). All mice entered the studies between 7 and 12 weeks of age.  
         [0027]    Cell Lines and tissue culture: A20 is a murine B-cell lymphoma cell line originally derived from a BALB/cAnN mouse (American Type Culture Collection, Manassas, Va.). The cells were passaged in vivo, expanded and frozen in aliquots. Single aliquots were thawed as needed for tumor challenge experiments. Mouse BALB/c-3T3 fibroblast were obtained from ATCC and cultured in DMEM medium (Mediatech, Herndon, Va.) supplemented with 100 g/mL penicillin, 100 μg/mL streptomycin, 2 mM glutamine (all from Gibco, Grand Island, N.Y.) and 10% fetal calf serum (HyClone, Logan, Utah). Cells were routinely tested for mycoplasma and found to be negative.  
       EXAMPLE 1  
       [0028]    Plasmid Construction:  
         [0029]    Murine CD20 cDNA was obtained by RT-PCR from C57/B6 mouse spleen and from A20 cells. The human counterpart was isolated from normal donor PBMC. Briefly, total RNA was extracted with Trizol reagent (InVitrogen, Carlsbad, Calif.) from 107 cells. The cDNA was synthesized using oligo dT primers and the DNAcycle kit (InVitrogen) as per the kit instructions. CD20 specific primers modified with appropriate restriction sites at the 5′ end (in both cases BamHI in the downstream primer and XbaI in the upstream primer) were as follows (all primers were synthesized by Operon, Alameda, Calif.): for the human cDNA the 5′ primer had the sequence 5′ATG GAT CCT GGA GGA AAT GCT GAG AG3′ (Seq. ID No. 1) and the 3′ primer 5′GGT CTA GAT GGT GCG TAT GTG CAG AG3′ (Seq. ID No. 2) and the resulting product measured 1158 bp and included 118 bp of 5′UTR, 894 bp of ORF and 130 bp of 3′UTR. The sequence of the 5′ and 3′ mouse primers were 5′AA G GAT CC A GTA CAC TGT AGC TAT CTT CAA GTA C3′ ((Seq. ID No. 3) and 5′CC T CTA GA A GAG TTT MG GAG CGA TCT C3′ (Seq. ID No. 4), respectively. The resulting PCR product measured 1008 bp with 127 bp of 5′ UTR, 876 bp of ORF and 5 bp of 3′UTR. PCR were performed for 35 cycles with the following conditions: 95° C. for 3 minutes, followed by 35 cycles of 95° C. for 30 seconds, 56° C. for 30 seconds and 68° C. for 1 minute and 30 seconds, followed by 7 minutes at 68° C. The PCR products were digested with BamHI and Xbal and subcloned into the mammalian expression vector pCR3 (InVitrogen) to create the plasmids pCR3-hCD20 and pCR3-mCD20 (FIGS. 1 a  and  1   b )(Seq. ID Nos. 5 and 6). The inserts were sequenced and compared to the mouse and human CD20 sequence found in the database. The mouse sequence was identical to the published sequence, while the human sequence had point mutations at position 117 of the ORF (C to T), at position 301 (T to C) and at position 324 (T to C), all of which were silent and likely represent normal polymorphism.  
       EXAMPLE 2  
       [0030]    For the construction of the minigenes, the inserts coding for the extracellular domain of CD20 were cloned by PCR into the plasmid pNERIS, in which the minigenes are expressed at the COOH-terminal of an endoplasmic reticulum (ER) insertion sequence derived from the adenovirus E3/19K glycoprotein, driven by the CMV promoter. The following primers were designed: the human downstream primer 5′CTA G CT GCA G TG TGC AGC GCC  TCC CAT TTT TTA AAA ATG GAG AGT C 3′ (Seq. ID No. 7) (Pstl site underlined, hCD20 sequence double underlined), and upstream primer 5′TG C TCG AG T CA A CAG TAT TGG GTA GAT GGG GAG3 ′ (Seq. ID No. 8)(Xhol site underlined, hCD20 sequence double underlined). The resulting product encodes the entire extracellular domain: KISHFLKMESLNFIR AHTPYINIYNCEPANPSEKNSPSTQYCYS (44 aa, (Seq. ID No. 9)) under the regulation of the CMV promoter. The downstream primer 5′CTA G CT GCA  GTG TGC AGC GCC  TCT CAT TTT TTA AAA ATG AGA AGA C 3′ (Seq. ID No. 10)(Pstl site underlined, hCD20 sequence double underlined), and the upstream primer 5′TG C TCG AG T CA A CAG TAC TGT GTA GAT GGG GAG 3′ (Seq. ID No.11)(XhoI site underlined, hCD20 sequence double underlined) were used to amplify the mouse extracellular domain with the sequence SHFLKMRRLELIQTSKPYVDIYDC EPSNSSEKNSPSTQYC (Seq. ID No.12). The PCR products were digested with Pstl and Xhol and subcloned into the pNERIS vector to create pNERIS-H8 and pNERIS-M8, respectively (FIGS. 1 c  and  1   d ). The plasmid were sequenced for accuracy (Seq. ID Nos. 13 and 14).  
       EXAMPLE 3  
       [0031]    Transfections: BALB/c-3T3 cells were grown to 50-80% confluency. Cells were transfected using the Lipofectamine reagent (InVitrogen) and 1 g each of the plasmids pCR3, pCR3-hCD20, pCR3-mCD20, pNERIS-H8 and pNERIS-M8, as per the manufacturer&#39;s instructions. Cell were selected 48 hours after transfection with 1 mg/mL G418 (Gibco). Expression of the transfected DNA was tested by RT-PCR, using the same CD20-specific primers described in the previous section.  
       EXAMPLE 4  
       [0032]    Gene gun immunization: DNA immunization was performed as previously described (Clinical Cancer Research 3:2191). Briefly, 100 μg of DNA from the plasmids described earlier was mixed with 50 mg of 0.95-2.6 μm diameter gold particles in the presence of 0.05 μM spermidine. CaCl2 to a final concentration of 400 mM was added drop wise and the DNA allowed to precipitate onto the gold particles for 10 minutes. The beads were then washed a minimum of three times with 100% ethanol and finally resuspended in7.2 mL 100% ethanol. This solution was instilled into 50 cm-long plastic Tefzel tubing, the ethanol was gently removed by aspiration and the gold particles were dried with a flow of nitrogen gas at 400 ml/min. The tube was then cut into 0.5 inches bullets. The gold-DNA complexes were injected into the shaved and depilated skin of anesthetized mice using a helium-driven gene gun (Powderject, Inc., Madison, Wis.). Four injections at 400 pounds/inch2 were delivered to each mouse, one per abdominal quadrant, for a total of 4 μg of DNA per mouse. Mice were immunized in this fashion weekly, for a total of five weeks for tumor challenge experiments and for a total of three weeks for ELISPOT assays.  
       EXAMPLE 5  
       [0033]    Tumor challenge and follow-up: Five days following the last of five immunizations, each mouse received 5×104 A20 cells by tail vein injection. The mice were housed in a tail vein illuminator device (Braintree Scientific, Braintree, Mass.) and given the cells resuspended in 200 μl of PBS. Two weeks after the tumor challenge, a baseline weight was taken for each mouse. Generally, symptoms of tumor take included the development of ascites, and consequently weight gain, and palpable abdominal masses arising from the liver and/or spleen. A few mice developed hind legs paralysis. Daily weights were recorded beginning at the third week after tumor challenge. Mice were sacrificed when overtly sick or when they gained 3 g of body weight.  
       EXAMPLE 6  
       [0034]    ELISPOT assays: Groups of five mice were immunized weekly×3. Five to 7 days after the last immunization, mice were sacrificed and the inguinal lymph nodes were isolated and pooled. A single cell suspension was obtained by nylon mesh filtering the disrupted lymph nodes. CD8+ T-cells were isolated by positive selection adding 20 μl of anti mouse CD8 immuno magnetic beads. The cell/beads suspension was run over MACS columns (Miltenyi Biotec Inc., Auburn, Calif.) and washed with MACS buffer (PBS with 0.5% BSA). Cells were eluted with 4 mL of MACS buffer, washed and resuspended in fresh medium. Fifty thousands CD8+T-cells were plated on each well in a 96-well plate coated overnight with 5 pg/ml anti-IFN antibody (Mabtech, Cincinnati, Ohio) in PBS. The cells were incubated at 37 C for 1 hour before overlaying 5×103 3T3 transfectants to each well. Each transfectant cell line was plated in triplicate. Cells were again incubated at 37 C for 20 hours. The next day the cells were washed with PBS/0.05% Tween (PBST) six times. Biotinylated anti-IFN antibody (Mabtech) diluted to 1 μg/mL in PBS/0.5% bovine serum albumin was added to the wells and incubated at 37 C for 2 hours. Plates were washed six times with PBST. Spots were developed with ABC reagent (Vectors Laboratories, Burlington, Calif.) for 30 minutes at room temperature. The reagent was removed with 6 washes with PBS and the spot developed with AEC in 0.5% dimethyl formamide. After 4 minutes the plates were washed with tap water and let air-dry. The plates were read with a Zeiss.  
       EXAMPLE 7  
       [0035]    Production of recombinant mouse CD20 (recCD20) fusion protein: the baculovirus expression system was used to produce a partially purified recCD20 protein for use as a xenoexpressed CD20 antigen. The mouse cDNA was amplified by PCR from pCR3-mCD20 using the same downstream primer described in the plasmid construct section, containing a BamHI site at the 5′ end, and the following upstream primer 5′CG G AAT TCA   GAG TTT AAG GAG CGA TCT C3 ′, containing an EcoRI (underlined, CD20 sequence double underlined). All reagents for the insect expression system were purchased from InVitrogen. The PCR product was digested with BamHI and EcoRI and subcloned into pBlueBacHis2 (b), a vector that expresses CD20 with a 6×His region and a Xpress epitope fused at the N-terminus, under the control of the polyhedrin promoter. The plasmid was transfected into Sf9 cells using the CelIFECTIN reagent together with linearized Bac-n-Blue viral DNA following the manufacturer instructions. The final result is recombinant virus with a functional lacZ gene for plaques screening and purification by plaque assay. Two positive recombinant plaques were isolated and propagated. High titer viral stock was prepared from one of the two clones (named B9). The infected cells lysate and the supernatant were tested at several time points by western blot analysis, using the anti-Xpress monoclonal antibody. The expected size of the fusion protein was around 40 kDa.  
       EXAMPLE 8  
       [0036]    Immunoprecipitation and Western blot: After five immunizations, serum was collected from each mouse by eye bleed and preserved at −20 C until needed. Five μL of serum were incubated with 10 μL of recombinant mCD20 overnight at 4 C. All samples were also prepared in the absence of recCD20 as a negative control for the secondary antibody. Ten μL of agarose beads-anti mouse IgG (Sigma., St. Louis, Mo.) were added to the mixture and incubated for 1 hour at 4 C. Beads were washed 5 times with 10 mM Tris-HCl, pH 7.5,150 mM NaCl, 5 mM EDTA and 1% NP-40 (TNEN), five times with {fraction (1/10)} strength TNEN and finally five times with distilled water. Proteins were analyzed by 4-20% gradient SDS-polyacrylamide gel electrophoresis under reducing conditions and transferred to Immobilon membrane (Millipore, Bedford, Mass.). The membranes were blocked overnight with 5% non-fat dry milk in TBS-0.05%Tween (TBST), washed with TBST then probed with anti-Xpress antibody diluted 1:5000 in TBST/3%BSA. After three more washes with TBST, the membranes were incubated with HRP-labeled anti mouse IgG (Sigma) diluted at 1:10,000. The membranes were washed again with TBST then developed with ECL reagent (Amersham Biosciences, Piscataway, N.J.) according to the manufacturer&#39;s instructions.  
                                                                                                           Sequence of PCR3-mCD20:                (Seq. ID No.6)                gcgcgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggag                   ttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgac               gtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggactatttacggtaaactycccacttgg               cagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcc               cagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggtt               ttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatggga               gtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtagg               cgtgtacggtggyaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcyaaattaa               tacgactcactatagggagacccaagcttggtaccgagctcggatccagtacactgtagctatcttcaagtacttgagat               agaagaggccaactgatctcagctgtgagtggctaatttggcccttaagccttgyagccttggagccttggagacccagg               cgtttgaaaactcaatgagtggacctttcccagcagagcctacaaaaggtcccctcgccatgcaacctgctccaaaagtg               aacctcaaaaggacatcttcactggtgggccccacacaaagcttcttcatgagggaatcaaaggctttgggggctgtcca               aatcatgaatggcctcttccatattaccctggggggactgctgatgatccccacaggggtcttcgcacccatctgtttga               gtgtatggtaccctctctggggaggcattatgtacattatttcaggatcactcctggcagctgcagcagaaaaaacctcc               aggaagagtttggtcaaagcaaaagtgataatgagctctctaagcctctttgctgccatttctgyaataattctttcaat               catggacatacttaacatgacactttctcattttttaaaaatgagaagactggagcttattcaaacttccaagccgtatg               ttgatatctacgactgtgaaccatctaattcctcagagaaaaactccccatctacacaytactgtaacagcattcagtct               gtgttcttgggcattctgtcggcgatgctgatctctgccttcttccagaaacttgtgacagctggtattgtggagaatga               gtggaaaagaatgtgtaccagatccaaatctaatgtggttctgctgtcagctygagaaaaaaatgagcagacgattaaaa               tgaaagaagaaatcattgagctaagtggagtatcttcccaaccaaagaatgaagaggaaattgaaattattccagtgcag               gaggaagaagaagaagaagcagaaataaattttccagcacctccccaagagcaggaatccttgccagtggaaaatgagat               cgctccttaaactcttctagagggccctattctatagtgtcacctaaatgctagagctcgctgatcagcctcgactgtgc               cttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctt               tcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacag               caagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaacca               gtggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccag               gaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaa               gtcagaggtggcgaaacccgacaggactataaagataccagycgtttccccctggaagctccctcgtgcgctctcctgtt               ccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtag               gtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgcct               tatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggatt               agcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatt               tggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctg               gtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttct               acggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcaccta               gatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaacctgaggctatggcagggcctgccg               ccccgacgttggctgcgagccctgggccttcacccgaacttggggggtggggtggggaaaaggaagaaacgcgggcgtat               tggccccaatggggtctcggtggggtatcgacagagtgccagccctgggaccgaaccccgcgtttatgaacaaacgaccc               aacaccgtgcgttttattctgtctttttattgccgtcatagcgcgggttccttccgttattgtctccttccgtgtttcag               ttagcctccccctagggtgggcgaagaactccagcatgagatccccgcgctggaggatcatccagccggcgtcccggaaa               acgattccgaagcccaacctttcatagaaggcggcggtggaatcgaaatctcgtgatggcaggttgggcgtcgcttggtc               ggtcatttcgaaccccagagtcccgctcagaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatcgggagc               ggcgataccgtaaagcacgaggaagcggtcagcccattcgccgccaagctcttcagcaatatcacgggtagccaacgcta               tgtcctgatagcggtccgccacacccagccggccacagtcgatgaatccagaaaagcggccattttccaccatgatattc               ggcaagcaggcatcgccatgggtcacgacgagatcctcgccgtcgggcatgctcgccttgagcctggcgaacagttcggc               tggcgcgagcccctgatgctcttgatcatcctgatcgacaagaccggcttccatccgagtacgtgctcgctcgatgcgat               gtttcgcttggtggtcgaatgggcaggtagccggatcaagcgtatgcagccgccgcattgcatcagccatgatggatact               ttctcggcaggagcaaggtgagatgacaggagatcctyccccggcacttcgcccaatagcagccaytcccttcccgcttc               agtgacaacgtcgagcacagctgcgcaaggaacgcccgtcgtggccagccacgatagccgcgctgcctcgtcttgcagtt               cattcagggcaccggacaggtcggtcttgacaaaaagaaccgggcgcccctgcgctgacagccgyaacacggcggcatca               gagcagccgattgtctgttgtgcccagtcatagccgaatagcctctccacccaagcggccggagaacctgcgtgcaatcc               atcttgttcaatcatgcgaaacgatcctcatcctgtctcttgatcgatctttgcaaaagcctaggcctccaaaaaagcct               cctcactacttctggaatagctcagaggccgaggcggcctcggcctctgcataaataaaaaaaattagtcagccatgggg               cggagaatgggcggaactgggcggagttaggggcgggatgggcggagttaggggcgggactatggttgctgactaattga               gatgcatyctttgcatacttctgcctgctggggagcctggggactttccacacctggttgctgactaattgagatgcatg               ctttgcatacttctgcctgctggggagcctggggactttccacaccctaactgacacacattccacagctggttctttcc               gcctcaggactcttcctttttcaataaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaa               tcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacg               atacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagc               aataaaccagccayccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgtt               gccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtca               cgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaa               aaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcag               cactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctga               gaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaa               agtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaac               ccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaat               gccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcattta               tcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttc               cccgaaaagtgccacctgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgct               acacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtca               agctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtg               atggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtgga               ctcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggc               ctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttac               Sequence of pCR3-hCD20:            (Seq. ID No.5)                gcgcgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggag                   ttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgac               gtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggactatttacggtaaactgcccacttgg               cagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcc               cagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggtt               ttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatggga               gtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtagg               cgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaa               tacgactcactatagggagacccaagcttggtaccgagctcggatccaagcattcagatgcatgacacaaggtaagactg               ccaaaaatcttgttcttgctctcctcattttgttatttgttttatttttaggagttttgagagcaaaatgacaacaccca               gaaattcagtaaatgggactttcccggcagagccaatgaaaggccctattgctatgcaatctggtccaaaaccactcttc               aggaggatgtcttcactggtgggccccacgcaaagcttcttcatgagggaatctaagactttgggggctgtccagattat               gaatgggctcttccacattgccctggggggtcttctgatgatcccagcagggatctatgcacccatctgtgtgactgtgt               ggtaccctctctggggaggcattatgtatattatttccggatcactcttggcagcaacggagaaaaactctaggaagtgt               ttggtcaaaggaaaaatgataatgaattcattgagcctctttgctgccatttctggaatgattctttcaatcatggacat               acttaatattaaaatttcccattttttaaaaatggagagtctgaattttattagagctcacacaccatatattaacatat               acaactgtgaaccagctaatccctctgagaaaaactccccatctacccaatactgttacagcatacaatctctgttcttg               ggcattttgtcagtgatgctgatctttgccttcttccaggaacttgtaatagctggcatcgttgagaatgaatggaaaag               aacgtgctccagacccaaatctaacatagttctcctgtcagcagaagaaaaaaaagaacagactattgaaataaaagaag               aagtggttgggctaactgaaacatcttcccaaccaaagaatgaagaagacattgaaattattccaatccaagaagaggaa               gaagaagaaacagagacgaactttccagaacctccccaagatcaggaatcctcaccaatagaaaatgacagctctcctta               agtgatttcttctgttttctgtttccttttttaaacattagtgttcatagcttccaagagacaggtctagagggccctat               tctatagtgtcacctaaatgctagagctcgctyatcagcctcgactgtgccttctagttgccagccatctgttgtttgcc               cctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcat               tgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcag               gcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagtggcggtaatacggttatccacagaatca               ggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtt               tttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactat               aaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtcc               gcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctc               caagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacc               cggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctaca               gagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttac               cttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagc               agattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaac               tcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaa               atcaatctaaagtatatatgagtaacctgaggctatggcagggcctgccgccccgacgttggctgcgagccctgggcctt               cacccgaacttggggggtggggtggggaaaaggaagaaacgcgggcgtattggccccaatggggtctcggtggggtatcg               acagagtgccagccctgggaccgaaccccgcgtttatgaacaaacgacccaacaccgtgcgttttattctgtctttttat               tgccgtcatagcgcgggttccttccggtattgtctccttccgtgtttcagttagcctccccctagggtgggcgaagaact               ccagcatgagatccccgcgctggaggatcatccagccggcgtcccggaaaacgattccgaagcccaacctttcatagaag               gcggcggtggaatcgaaatctcgtgatggcaggttgggcgtcgcttggtcggtcatttcgaaccccagagtcccgctcag               aagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatcgggagcggcgataccgtaaagcacgaggaagcggtc               agcccattcgccgccaagctcttcagcaatatcacgggtagccaacgctatgtcctgatagcggtccgccacacccagcc               ggccacagtcgatgaatccagaaaagcggccattttccaccatgatattcggcaagcaggcatcgccatgggtcacgacg               agatcctcgccgtcgggcatgctcgccttgagcctggcgaacagttcggctggcgcgagcccctgatgctcttgatcatc               ctgatcgacaagaccggcttccatccgagtacgtgctcgctcgatgcgatgtttcgcttggtggtcgaatgggcaggtag               ccggatcaagcgtatgcagccgccgcattgcatcagccatgatggatactttctcggcaggagcaaggtgagatgacagg               agatcatgccccggcacttcgcccaatagcagccagtcccttcccgcttcagtgacaacgtcgagcacagctgcgcaagg               aacgcccgtcgtggccagccacgatagccgcgctgcctcgtcttgcagttcattcagggcaccggacaggtcggtcttga               caaaaagaaccgggcgcccctgcgctgacagccggaacacggcggcatcagagcagccgattgtctgttgtgcccagtca               tagccgaatagcctctccacccaagaggccggagaacctgcgtgcaatccatcttgttcaatcatgcgaaacgatcctca               tcctgtctcttgatcgatctttgcaaaagcctaggcctccaaaaaagcctcctcactacttctggaatagctcagaggcc               gaggcggcctcggcctctgcataaataaaaaaaattagtcagccatggggcggagaatgggcggaactgggcggagttag               gggcgggatgggcggayttaggggcgggactatggttgctgactaattgagatgcatgctttgcatacttctgcctgctg               gggagcctygggactttccacacctggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcct               ggggactttccacaccctaactgacacacattccacagctggttctttccgcctcaggactcttcctttttcaataaatc               aatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtc               tatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagt               gctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcg               cagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccag               ttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagc               tccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgat               cgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccat               ccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctct               tgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggg               gcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcat               cttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacgg               aaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacat               atttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgcgccctgta               gcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcyccctagcgcccgctcct               ttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggtt               ccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgat               agacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaac               cctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaaca               aaaatttaacgcgaattttaacaaaatattaacgcttacaatttac               Sequence of pNERIS-M8:            (Seq ID No.14)                gcgcgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggag                   ttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgac               gtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggactatttacggtaaactgcccacttgg               cagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcc               cagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggtt               ttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatggga               gtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtagg               cgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaa               tacgactcactatagggagacccaagcttggtaccgagctcggatccaccatgagatacatgatcctgggcctgctggcc               ctggctgcagtgtgcagcgcctctcattttttaaaaatgagaagactggagcttattcaaacttccaagccgtatgttga               tatctacgactgtgaaccatctaattcctcagagaaaaactccccatctacacagtactgttctcgagcatgcatctaga               gggccctattctatagtgtcacctaaatgctagagctcgctgatcagcctcgactgtgccttctagttgccagccatctg               ttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaatt               gcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaaga               caatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagtggcggtaatacggttatcc               acagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgtt               gctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccga               caggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccgga               tacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggt               cgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttg               agtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggc               ggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaa               gccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgttt               gcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtgg               aacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatg               aagttttaaatcaatctaaagtatatatgagtaacctgaggctatggcagggcctgccgccccgacgttggctgcgagcc               ctgggccttcacccgaacttggggggtggggtggggaaaaggaagaaacgcgggcgtattggccccaatggggtctcggt               ggggtatcgacagagtgccagccctgggaccgaaccccgcgtttatgaacaaacgacccaacaccgtgcgttttattctg               tctttttattgccgtcatagcgcgggttccttccggtattgtctccttccgtgtttcagttagcctccccctagggtggg               cgaagaactccagcatgagatccccgcgctggaggatcatccagccggcgtcccggaaaacgattccgaagcccaacctt               tcatagaaggcggcggtggaatcgaaatctcgtgatggcaggttgggcgtcgcttggtcggtcatttcgaaccccagagt               cccgctcagaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatcgggagcggcgataccgtaaagcacgag               gaagcggtcagcccattcgccgccaagctcttcagcaatatcacgggtagccaacgctatgtcctgatagcggtccgcca               cacccagccggccacagtcgatgaatccagaaaagcggccattttccaccatgatattcggcaaycagycatcgccatgg               gtcacgacgagatcctcgccgtcgggcatgctcgccttgagcctggcgaacayttcggctggcgcgagcccctgatgctc               ttgatcatcctgatcgacaagaccggcttccatccgagtacgtgctcgctcgatgcgatgtttcgcttggtygtcgaatg               ggcaggtagccggatcaagcgtatgcagccgccgcattgcatcagccatgatggatactttctcggcaggagcaaggtga               gatgacaggagatcctgacccggcacttcgcccaatagcagccagtcccttcccgcttcagtgacaacgtcgagcacagc               tgcgcaaggaacgcccgtcgtggccagccacyatagccgcgctgcctcgtcttgcagttcattcagggcaccggacaggt               cggtcttgacaaaaagaaccgggcgcccctgcgctgacagccggaacacggcggcatcagagcagccgattgtctgttgt               gcccagtcatagccgaatagcctctccacccaagcggccggagaacctgcgtgcaatccatcttgttcaatcatgcgaaa               cgatcctcatcctgtctcttgatcgatctttgcaaaagcctaggcctccaaaaaagcctcctcactacttctggaatagc               tcagaggccgaggcggcctcggcctctgcataaataaaaaaaattagtcagccatggggcggagaatyggcggaactggg               cggagttaggggcgggatgggcggagttaggggcgggactatggttgctgactaattgagatgcatgctttgcatacttc               tgcctgctggggagcctggggactttcaacacctggttgctgactaattgagatgcatgctttgcatacttctgcctgct               ggggagcctggggactttccacaccctaactgacacacattccacagctggttctttccgcctcaggacecttccttttt               caataaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcag               cgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatct               ggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaag               ggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctayagtaagta               gttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggct               tcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcgg               tcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactg               tcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccg               agttgctcttgcccggcgtcaatacgggataataecgcgccacatagcagaactttaaaagtgctcatcattggaaaacg               ttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgat               cttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagg               gcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgag               cggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacg               cgccctgtagcggcgcattaagcgcggcyggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcg               cccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccc               tttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccat               cgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaaca               acactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagct               gatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttac               Sequence of pNERIS-H8:            (Seq. ID No.13)                gcgcgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggag                   ttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgac               gtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggactatttacggtaaactgcccacttgg               cagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggeattatgcc               cagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggtt               ttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatggga               gtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtagg               cgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaa               tacgactcactatagggagacccaagcttggtaccgagctcggatccaccatgagatacatgatcctgggcctgctggcc               ctggctgcagtgtgcagcgcctcccattttttaaaaatggagagtctgaattttattagayctcacacaccatatattaa               catatacaactgtgaaccagctaatccctctgagaaaaactccccatctacccaatactgttcatctcgagcatgcatct               agagggccctattctatagtgtcacctaaatgctagagctcgctgatcagcctcgactgtgccttctagttgccagccat               ctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaa               attgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattggga               agacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagtggcggtaatacggtta               tccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgc               gttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacc               cgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttacc               ggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgta               ggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtc               ttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgta               ggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcyctctgct               gaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttg               tttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcag               tggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaa               atgaagttttaaatcaatctaaagtatatatgagtaacctgaggctatggcagggcctgccgccccgacgttggctgcga               gccctgggccttcacccgaacttggggggtggggtggggaaaaggaagaaacgcgggcgtattggccccaatggggtctc               ggtggggtatcgacagagtgccagccctgggaccgaaccccgcgtttatgaacaaacgacccaacaccgtgcgttttatt               ctgtctttttattgccgtcatagcgcgggttccttccggtattgtctccttccgtgtttcagttagcctccccctagggt               gggcgaagaactccagcatgagatccccgcgctggaggatcatccagccggcgtcccggaaaacgattccgaagcccaac               ctttcatagaaggcggcggtggaatcgaaatctcgtgatggcaggttgggcgtcgcttggtcggtcatttcgaaccccag               agtcccgctcagaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatcgggagcggcgataccgtaaagcac               gaggaagcggtcagcccattcgccgccaagctcttcagcaatatcacgggtagccaacgctatgtcctgatagcggtccg               ccacacccagccggccacagtcgatgaatccagaaaagcggccattttccaccatgatattcggcaagcaggcatcycca               tgggtcacgacgagatcctcgccgtcgggcatgctcgccttgagcctggcgaacagttcggctggcgcgaycccctgatg               ctcttgatcatcctgatcgacaagaccggcttccatccgagtacgtgctcgctcgatgcgatgtttcgcttggtggtcga               atgggcaggtagccggatcaagcgtatgcagccgccgcattgcatcagccatgatggatactttctcggcaggagcaagg               tgagatgacaggagatcctgccccggcacttcgcccaatagcagccagtcccttcccgcttcagtgacaacgtcgagcac               agctgcgcaaggaacgcccgtcgtggccagccacgatagccgcgctgcctcgtcttgcagttcattcagggcaccggaca               ggtcggtcttgacaaaaagaaccgggcgcccctgcgctgacagccggaacaaggcggcatcagagcagccgattgtctgt               tgtgcccagtcatagccgaatagcctctccacccaagcggccggagaacctgcgtgcaatccatcttgttcaatcatgcg               aaacgatcctcatcctgtctcttgatcgatctttgcaaaagcctaggcctccaaaaaagcctcctcactacttctggaat               agctcagaggccgaggcggcctcggcctctgcataaataaaaaaaattagtcagccatggggcggagaatgggcggaact               gggcggagttaggggcgggatgggcggagttaggggcgggactatggttgctgactaattgagatgcatgctttgcatac               ttctgcctgctggggagcctggggactttccacacctggttgctgactaattgagatgcatgctttgcatacttctgcct               gctggggagcctggggactttccacaccctaactgacacacattccacagctggttctttccgcctcaggactcttcctt               tttcaataaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatct               cagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttacca               tctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccgg               aagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaa               gtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatg               gcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctcctt               cggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctctta               ctgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcga               ccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaa               acgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaact               gatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatyccgcaaaaaagggaata               agggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcat               gagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctg               acgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgcccta               gcgcccgctcctttcgctttcttcccttcctttctcgcgacgttcgccggctttccccgtcaagctctaaatcgggggct               ccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggc               catcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactgga               acaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatga               gctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttac