# Patent

## Title
B7-1-PE40KDEL exotoxin fusion gene-based DNA vaccine and the use thereof

## Abstract

The present invention relates to the fields of immunology and molecular biology and related to a B7-1-PE40KDEL exotoxin fusion gene-based DNA vaccine and the use thereof. Specifically, the DNA vaccine contains a recombinant expression vector, and the vector contains exotoxin fusion gene B7-1-PE40KDEL, which is effectively ligated into selected eukaryotic expression vectors, such as pcDNA3.1/Zeo(+), pWLNEO, pSV2CAT, pOG44, pXT1, pSG, pSVK3, pBPV, pMSG, pSVL, and adenovirus. The invention also relates to the exotoxin fusion gene B7-1-PE40KDEL, the encoded exotoxin fusion protein, a recombinant expression vector that contains the exotoxin fusion gene, and compositions that contain the recombinant expression vector. The DNA vaccine in this invention has a good effect on the treatment or prevention of allogeneic tissue/organ transplant rejection and hematopoietic stem cell transplantation rejection such as GVHD.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
The present application is a 35 U.S.C. §371 national stage patent application of International patent application PCT/CN2011/079862, filed on Sep. 20, 2011, published as WO/2012/094905 on Jul. 19, 2012, the text of which is incorporated by reference, and claims the benefit of the filing date of Chinese application no. 201110023985.6, filed on Jan. 14, 2011, the text of which is also incorporated by reference.
FIELD OF THE INVENTION
The present invention relates to the fields of immunology and molecular biology and is related to a B7-1-PE40KDEL exotoxin fusion gene-based DNA vaccine and the use thereof. The invention also relates to a B7-1-PE40KDEL exotoxin fusion gene, an exotoxin fusion protein encoded by the fusion gene, a recombinant expression vector containing the fusion gene, and a composition that contain the recombinant expression vector.
BACKGROUND OF THE INVENTION
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) and solid organ transplantation have been widely used in the treatment of hematologic malignancies, certain genetic diseases, acute severe radiation sickness, and a variety of solid organ failures and malignancies. Due to the differences in the major histocompatibility antigen (MHC) between donors and recipients, along with many other known and unknown factors such as the minor histocompatibility antigen (mHA), tissue-specific antigens, and non-immunological factors, host-versus-graft diseases (HVGDs) and graft-versus-host diseases (GVHDs or GVHRs) are inevitable and are one of the most important and fundamental causes of allogeneic tissue graft failure and graft chronic dysfunction (GCD). With the increasing number of patients in urgent need of organ transplants, the lack of donors is increasingly prominent. Both now and in the future, transplant cases in which the MHCs are not fully compatible or are only semi-compatible will become mainstream and constitute the new direction of hematopoietic stem cell transplantation and organ transplantation. The traditional methods for the prevention and treatment of GVHD and HVGD are based on the destruction of the entire immune function in transplant recipients, even at the cost of the loss of graft-versus-leukemia (GVL) and anti-inflammatory functions, implemented by the use of a variety of high-dose immunosuppressants or the removal of all T cells from the graft. There is no doubt that the fundamental method behind any new strategy to prevent the occurrence and development of GVHD and HVGD depends on whether the initial step of T cell activation can be effectively interfered with or inhibited to induce specific immune tolerance, thereby fundamentally curbing the occurrence and development of a “cytokine storm”. Therefore, an ideal and efficient allo-HSCT and organ transplantation should amend or modify the initial reaction of T cells to allogeneic antigens rather than removing all T cells from the body of the transplant recipient or the donor graft. That is, the induction of recipient or donor-specific immune tolerance should target the elimination or inhibition of the recipient or donor T-cell reaction to allogeneic antigens but still retain the normal reaction of T cells to other antigens.
It has been proven that the recipient and donor T cells play a dual role in hematopoietic stem cell transplantation or organ transplantation. In addition to contributing to the engraftment of hematopoietic stem cells or organs and control conditional infections and anti-leukemia effects, they can also be the root cause of GVHD or HVGD. This immune response is accomplished through recognition and activation by T cells. T cell activation requires two signals: T cell receptor signal and co-stimulation signal of cluster of differentiation 28 (CD28). The first signal is regulated by the second signal, resulting in T cell activation, partial activation or anergy. Therefore, inhibiting T cell activation by blocking either the T cell receptor signal or the CD28-mediated co-stimulatory signal is an important strategy for the prevention of HVGD and acute GVHD (aGVHD). The cytotoxic T-lymphocyte antigen 4-Ig (CTLA4-Ig) or B7 antibody-induced specific immune tolerance is primarily used to achieve the goal of preventing HVGD and aGVHD and has been proven effective in a large number of in vivo and in vitro animal experiments. However, the shortcomings of this method are that the duration of the induced immune tolerance is short and that the induced incompetent T cells can be reactivated by other signaling pathways, leading to treatment failure. In addition, the monoclonal antibodies used for blocking are mostly murine, and their immunogenicity will affect the treatment's efficacy.
Therefore, a DNA vaccine with good immune tolerance and therapeutic efficacy for the treatment or the prevention of allogeneic tissue/organ transplant rejection such as GVHD is urgently needed in this field.
SUMMARY OF THE INVENTION
After numerous experiments and creative work, the inventors have discovered a B7-1-PE40KDEL exotoxin fusion gene and, surprisingly, found that the recombinant expression vector pcDNA3.1/Zeo(+)-B7-1-PE40KDEL that is transfected into eukaryotic cells is transcribed, translated, post-translationally modified, and then secreted into the extracellular space. The vector pcDNA3.1/Zeo(+)-B7-1-PE40KDEL can be efficiently expressed in eukaryotic cells, and the expression product has good targeting immunosuppressive activity. The inventors have also found that the pcDNA3.1/Zeo(+)-B7-1-PE40KDEL exotoxin fusion gene-based DNA vaccine can be effective in the prevention and treatment in murine aGVHD model. Therefore, the following invention is provided.
One aspect of the invention relates to a B7-1-PE40KDEL exotoxin fusion gene, and its nucleotide sequence is shown as SEQ ID NO: 1 or SEQ ID NO: 2.
The determination of the B7-1-PE40KDEL sequence and the open reading frame are analyzed as follows:
The nucleotide sequence of SEQ ID NO: 1 (1850 base pairs [bp]):













60



TCTGGGTTCC AGGTTCCACT GGTGACGTTA TCCACGTGAC CAAGGAAGTG AAAGAAGTAG
120


 


CAACGCTGTC CTGTGGTCAC AATGTTTCTG TTGAAGAGCC GGCACAAACT CGCATCTACT
180


 


GGCAAAAGGA GAAGAAAATG GTGCTGACTA TGATGTCTGG GGACATGAAT ATATGGCCCG
240


 


AGTACAAGAA CCGGACCATC TTTGATATTA CTAATAACCT CTCCATTGTG ATCCTGGCTC
300


 


TGCGCCCATC TGACGAGGGC ACATACGAGT GTGTTGTTCT GAAGTATGAA AAAGACGCTT
360


 


TCAAGCGGGA ACACCTGGCT GAAGTGACGT TATCAGTCAA AGCTGACTTC CCTACACCTA
420


 


GTATATCTGA CTTTGAAATT CCAACTTCTA ATATTAGAAG GATAATTTGC TCAACCTCTG
480


 


GAGGTTTTCC AGAGCCTCAC CTCTCCTGGT TGGAAAATGG AGAAGAATTA AGTGCCATCA
540


 


ACACAACAGT TTCCCAAGAT CCTGAAACTG AGCTCTATGC TGTTAGCAGC AAACTGGATT
600


 


TCAATATGAC AACCAACCAC AGCTTCATGT GTCTCATCAA GTATGGACAT TTAAGAGTGA
660


 


ATCAGACCTT CAACTGGAAT ACAACCAAGC AAGAGCATTT TCCTGATAAC GGTGGCGGCG
720


 


GATCTGGAGG CGGTGGAAGC GGTGGTGGCT CGGGCGGTGG TGGGTCGGGC GGCAGCCTGG
780


 


CCGCGCTGAC CGCGCACCAG GCTTGCCACC TGCCGCTGGA GACTTCCACC CGTCATCGCC
840


 


AGCCGCGCGG CTGGGAACAA CTGGAGCAGT GCGGCTATCC GGTGCAGCGG CTGGTCGCCC
900


 


TCTACCTGGC GGCGCGGCTG TCGTGGAACC AGGTCGACCA GGTGATCCGC AACGCCCTGG
960


 


CCAGCCCCGG CAGCGGCGGC GACCTGGGCG AAGCGATCCG CGAGCAGCCG GAGCAGGCCC
1020


 


GTCTTGCCCT GACCCTGGCC GCCGCCGAGA GCGAGCGCTT CGTCCGGCAG GGCACCGGCA
1080


 


ACGACGAGGC CGGCGCGGCC AACGCCGACG TGGTGAGCCT GACCTGCCCG GTCGCCGCCG
1140


 


GTGAATGCGC GGGCCCGGCG GACAGCGGCG ACGCCCTGCT GGAGCGCAAC TATCCCACTG
1200


 


GCGCGGAGTT CCTCGGCGAC GGCGGCGACG TCAGCTTCAG CACCCGCGGC ACGCAGAACT
1260


 


GGACGGTGGA GCGGCTGCTC CAGGCGCACC GCCAACTGGA GGAGCGCGGC TATGTGTTCG
1320


 


TCGGCTACCA CGGCACCTTC CTCGAAGCGT CGCAAAGCAT CGTCTTCGGC GGGGTGCGCG
1380


 


CGCGCAACCA GGACCTCGAC GCGATCTGGC GCGGTTTCTA TATCGCCGGC GATCCGGCGC
1440


 


TGGCCTACGG CTACGCCCAG GACCAGGAAC CCGACGCACG CGGCCGGATC CGCAACGGTG
1500


 


CCCTGCTGCG GGTCTATGTG CCGCGCTCGA GCCTGCCGGG CTTCTACCGC ACCAGCCTGA
1560


 


CCCTGGCCGC GCCGGAGGCG GCGGGCGAGG TCGAACGGCT GATCGGCCAT CCGCTGCCGC
1620


 


TGCGCCTGGA CGCCATCACC GGCCCCGAGG AGGAAGGCGG GCGCCTGGAG ACCATTCTCG
1680


 


GCTGGCCGCT GGCCGAGCGC ACCGTGGTGA TTCCCTCGGC GATCCCCACC GACCCGCGCA
1740


 


ACATCGGCGG CGACCTCGAC CCGTCCAGCA TCCCCGACAA GGAACAGGCG ATCAGCGCCC
1800


 






1850






In the above sequence, the underlined parts are Kpn I restriction sites, and the framed parts are the start codon and stop codon. The open reading frame of the above sequence SEQ ID NO: 1 is SEQ ID NO: 2 (1827 bp).
Another aspect of the invention relates to the exotoxin fusion protein (SEQ ID NO: 3) encoded by the exotoxin fusion gene (SEQ ID NO: 1 or SEQ ID NO: 2).
The amino acid sequence of SEQ ID NO: 3 is shown as follows: (608 amino acids, the underlined part is the signal peptide sequence).








Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro



1               5                   10                  15


 


Gly Ser Thr Gly Asp Val Ile His Val Thr Lys Glu Val Lys Glu Val


            20                  25                  30


 


Ala Thr Leu Ser Cys Gly His Asn Val Ser Val Glu Glu Pro Ala Gln


        35                  40                  45


 


Thr Arg Ile Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met


    50                  55                  60


 


Ser Gly Asp Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe


65                  70                  75                  80


 


Asp Ile Thr Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser


                85                  90                  95


 


Asp Glu Gly Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala


            100                 105                 110


 


Phe Lys Arg Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp


        115                 120                 125


 


Phe Pro Thr Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile


    130                 135                 140


 


Arg Arg Ile Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu


145                 150                 155                 160


 


Ser Trp Leu Glu Asn Gly Glu Glu Leu Ser Ala Ile Asn Thr Thr Val


                165                 170                 175


 


Ser Gln Asp Pro Glu Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp


            180                 185                 190


 


Phe Asn Met Thr Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly


        195                 200                 205


 


His Leu Arg Val Asn Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu


    210                 215                 220


 


His Phe Pro Asp Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly


225                 230                 235                 240


 


Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser Leu Ala Ala Leu Thr


                245                 250                 255


 


Ala His Gln Ala Cys His Leu Pro Leu Glu Thr Ser Thr Arg His Arg


            260                 265                 270


 


Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln


        275                 280                 285


 


Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val


    290                 295                 300


 


Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp








305                 310                 315                 320



 


Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu


                325                 330                 335


 


Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly


            340                 345                 350


 


Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp Val Val Ser Leu Thr Cys


        355                 360                 365


 


Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ata


    370                 375                 380


 


Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly


385                 390                 395                 400


 


Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu


                405                 410                 415


 


Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe


            420                 425                 430


 


Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe


        435                 440                 445


 


Gly Gly Val Arg Ala Arg Asn Gln Asp Leu Asp Ala Ile Trp Arg Gly


    450                 455                 460


 


Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp


465                 470                 475                 480


 


Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg


                485                 490                 495


 


Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu


            500                 505                 510


 


Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly


        515                 520                 525


 


His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu


    530                 535                 540


 


Gly Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr


545                 550                 555                 560


 


Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Ile Gly Gly


                565                 570                 575


 


Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala


            580                 585                 590


 


Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro Lys Asp Glu Leu


    595                 600                 605






Another aspect of the invention relates to a recombinant expression vector containing the B7-1-PE40KDEL exotoxin fusion gene that can be effectively fused with selected eukaryotic expression vectors, such as pcDNA3.1/Zeo(+), pWLNEO, pSV2CAT, pOG44, pXT1, pSG, pSVK3, pBPV, pMSG, pSVL, and adenoviruses.
In one aspect of the invention, the recombinant expression vector consists of the B7-1-PE40KDEL gene and pcDNA3.1/Zeo(+) vector.
Another aspect of the invention relates to a composition containing any of the recombinant expression vectors in the invention.
The invention also relates to a DNA vaccine for the treatment or prevention of GVHD, which includes any of the recombinant expression vectors in the invention.
In one aspect of the invention, DNA vaccine described herein also includes pharmaceutically acceptable immune adjuvants.
In another aspect of the invention, DNA vaccine described herein is used for immunization via injection or mucosal or gene gun transfer. Specifically, DNA vaccine is used for immunization via at least one of the following methods: intravenous injection, intraarterial injection, intramuscular injection, subcutaneous injection, organ injection, pleural injection, and intraperitoneal injection.
In another aspect of the invention, the DNA vaccine described herein can be in the form of either an aqueous solution or a reconstituted freeze-dried powder used for injection or administration via the mucosae.
Another aspect of the invention relates to a method of DNA vaccine preparation, including the procedures to effectively fuse the B7-1-PE40 exotoxin fusion gene containing the SEQ ID NO: 1 or 2 nucleotide sequence and the pcDNA3.1/Zeo(+) vector.
The methods of DNA vaccine preparation described herein according to the invention include the following steps:
1) An upstream primer (P1) that contains the signal peptide and Kpn I restriction sites and a downstream primer (P2) that contains the Xba I restriction sites are designed. The primer sequences described are shown as SEQ ID NO: 4 and SEQ ID NO: 5.
2) The prokaryotic expression vector pGEMT-B7-1-PE40KDEL is used as the template for polymerase chain reaction (PCR) amplification using high-fidelity Pfu DNA polymerase.
3) PCR products are recovered. The recovered PCR product and the pcDNA3.1/Zeo(+) vector are double-digested with Kpn I+Xba 1 restriction enzymes, and the digested products are recovered after electrophoresis.
4) The digested products are recovered.
5) The double-digested PCR product and the pcDNA3.1/Zeo(+) vector are ligated.
6) Ampicillin-resistant bacterial clones are selected, and plasmids are extracted and identified by double digestion with restriction enzymes. The correct bacterial clones are cryopreserved.
7) The bacterial clone is cultured, and plasmids for DNA vaccine are extracted and purified.
8) The obtained plasmids are mixed with pharmaceutically acceptable immune adjuvants.
Another aspect of the invention relates to the exotoxin fusion gene or the application of the recombinant expression vector in the preparation of a DNA vaccine for the treatment or prevention of GVHD.
An additional aspect of the invention relates to the methods for the treatment or prevention of GVHD, including the steps of administering the effective treatment or prevention doses of a DNA vaccine to the subject.
The daily dose can be administered to the subject all at once or can also be administered in two, three, four, or more smaller doses at appropriate intervals over the course of a day. Smaller doses described herein may be prepared as unit doses, for example, each unit dose contains the dose that corresponds to the total daily dose divided by the number of appropriate doses. Additionally, DNA vaccine can also be administered at a certain interval: once a day, once every two days, once a week, once a month, once every two months, once every three months, once every six months, once a year, or once every two years.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. The schematic representation of construction of eukaryotic expression vector pcDNA3.1/B7-1-PE40KDEL, in which S indicates the signal peptide sequence.
FIG. 2. Agarose gel electrophoresis analysis of B7-1-PE40KDEL PCR products. Lane 1, DNA marker; Lane 2, B7-1-PE40KDEL PCR products.
FIG. 3. Agarose gel electrophoresis analysis of double-digested recombinant plasmid pcDNA3.1/B7-1-PE40KDEL was double-digested with Kpn I+Xba I restriction enzymes. Lane 1, DNA marker; Lane 2, Xba I-digested recombinant plasmid; Lane 3, Kpn I+Xba I double-digested recombinant plasmid.
FIG. 4. Agarose gel electrophoresis analysis of reverse transcriptase-PCR (RT-PCR) products of transfected CHO-K1-RPE.40 cells. Lane 1, DNA marker; Lane 2, B7-1-PE40KDEL amplification products in pcDNA3.1/B7-1-PE40KDEL-transfected CHO-K1-RPE.40 cells; Lane 3, β-actin PCR products; Lane 4, B7-1-PE40KDEL amplification products in pcDNA3.1 empty vector-transfected CHO-K1-RPE.40 cells; Lane 5, B7-1-PE40KDEL amplification products in untransfected CHO-K1-RPE.40 cells.
FIG. 5. Western blot analysis of B7-1-PE40KDEL fusion protein secreted from transfected eukaryotic cells. Lane 1, transfected with pcDNA3.1/Zeo(+)-B7-1-PE40KDEL; Lane 2, transfected with pcDNA3.1/Zeo(+) empty vector.
FIG. 6. Relative quantitative results of Pseudomonas exotoxin A (PEA) expression in stably transfected CHO-K1-RPE.40 with Zeo screening. A, real-time quantitative PCR; B, amplification curve; C, solubility curve.
FIG. 7. Standard curve of the measurement of PEA concentration.
FIG. 8. Comparison of the selective inhibitory activity of eukaryotic expression products B7-1-PE40KDEL.
FIG. 9. Agarose gel electrophoresis analysis of the serum expression level of pcDNA3.1/B7-1-PE40KDEL plasmid after intramuscular injection.
FIG. 10. Relative quantitative PE40KDELmRNA in blood.
FIGS. 11A through L. Representative photos of flow cytometric analysis of CD3+CD28+T counts in peripheral blood.
FIG. 12. The reduction of peripheral blood CD28+T cells after an intramuscular injection of B7-1-PE40KDEL DNA vaccines.
FIG. 13. Standard curve of the measurement of the serum Anti-PEA IgG antibody concentration.
FIG. 14. Typical GVHD symptoms in recipient mice. Typical hunched posture (A) and epilation (B).
FIG. 15. The changes in mice body weight in all groups after transplantation. A, aGVHD model group; B, Bone marrow transplantation group; C, Spleen cell infusion group; D, Radiation group alone.
FIG. 16. Chimerism analysis after transplantation: electrophoresis of the PCR products of sex-determining region Y (sry gene in Y chromosome). Lane 1, DNA marker; Lane 2, sry gene amplification in aGVHD model group; Lane 3, sry gene amplification in bone marrow transplantation group; Lane 4, sry gene amplification in spleen cell infusion group; Lane 5, sry gene amplification in radiation group alone.
FIG. 17. Histopathological examinations of aGVHD mice (hematoxylin and eosin [HE] staining×100). A, Small intestine; B, Liver; C, Spleen; D, Skin.
FIG. 18. The survival rates of GVHD mice in all groups after transplantation.
FIG. 19. The changes in body weight in GVHD mice in all groups after transplantation.
FIG. 20. The change in white blood cell (WBC) counts in aGVHD mice in all groups after transplantation.
FIG. 21. Histopathological changes in the small intestines of aGVHD mice in all groups after treatment with B7-1-PE40KDEL DNA vaccine and B7-2-PE40KDEL DNA vaccines (HE staining, 40×): A group (B7-1-PE40KDEL DNA vaccine), B group (B7-2-PE40KDEL DNA vaccine), C group (B7-1-PE40KDEL DNA vaccine+B7-2-PE40KDEL DNA vaccine), D group (empty vector), E group (cyclosporin A [CsA]+methotrexate [MTX]) and F group (untreated aGVHD group).
FIG. 22. Histopathological changes in the skin of aGVHD mice in all groups after treatment with B7-1-PE40KDEL DNA vaccine and B7-2-PE40KDEL DNA vaccines (HE staining, 40×): A group (B7-1-PE40KDEL DNA vaccine), B group (B7-2-PE40KDEL DNA vaccines), C group (B7-1-PE40KDEL+B7-2-PE40KDEL DNA vaccines), D group (empty vector), E group (CsA+MTX) and F group (untreated aGVHD group).
FIG. 23. Histopathological changes in the livers of aGVHD mice in all groups after treatment with B7-1-PE40KDEL DNA vaccine and B7-2-PE40KDEL DNA vaccine (HE staining, 40×): A group (B7-1-PE40KDEL DNA vaccine), B group (B7-2-PE40KDEL DNA vaccine), C group (B7-1-PE40KDEL DNA vaccine+B7-2-PE40KDEL DNA vaccine), D group (empty vector), E group (CsA+MTX) and F group (untreated aGVHD group).
FIG. 24. The survival curves and median survival times of aGVHD mice in all groups after treatment with B7-1-PE40KDEL and B7-2-PE40KDEL DNA vaccines.
FIG. 25. Chimerism analysis in all groups after transplantation: Electrophoresis of the sry gene (Y chromosome) PCR products. Lane 1, DNA marker; Lane 2, sry gene amplification in B7-1-PE40KDEL DNA vaccine group; Lane 3, sry gene amplification in B7-2-PE40KDEL DNA vaccine group; Lane 4, sry gene amplification in B7-1-PE40KDEL+B7-2-PE40KDEL DNA vaccines group; Lane 5, sry gene amplification in the pcDNA3.1 infusion group; Lane 6, sry gene amplification in CsA+MTX group; Lane 7, sry gene amplification in aGVHD model group.
FIG. 26. The effect of DNA vaccine on CD4+/CD8+ ratio of peripheral blood in aGVHD mice.
FIGS. 27A through N. Representative photos of flow cytometric analysis of CD3+CD4+T/CD3+CD8+T ratios of peripheral blood in all groups.
FIG. 28. The effect of DNA vaccine on CD4+CD25+Treg/CD4+T ratios of peripheral blood in all groups.
FIGS. 29A through G. Representative photos of flow cytometric analysis of CD4+CD25+Treg ratios of peripheral blood in all groups.
FIG. 30. The effect of DNA vaccine on CD8+CD28−Ts ratios of peripheral blood in all groups.
FIGS. 31A through G. Representative photos of flow cytometric analysis of CD8+CD28−Ts ratios of peripheral blood in all groups.



DETAILED DESCRIPTION OF THE INVENTION
The embodiment of the invention is described in detail below using examples. A person skilled in the art should note that the following examples are only used to explain the invention and should not be regarded as the limits of the invention scope. For the examples in which the specific techniques or conditions are not noted, the techniques and conditions are based on those described in the literature in this field (e.g., J. Sambrook et al., [translated by Peitang Huang et al.], Laboratory Manual of Molecular Cloning, Third Edition, Science Press) or the product manual. If the manufacturers of reagents or instruments are not noted, the reagents or instruments are all conventional products that can be purchased commercially.
Example 1
Construction of Eukaryotic Expression Vector pcDNA3.1/B7-1-PE40KDEL
1. Materials and Methods
1.1 Plasmids, Cells, and Main Reagents
The prokaryotic expression vector pGEMT-B7-1-PE40KDEL (2003.3.3) was constructed by our group, the strain contains said vector named as Escherichia coli DHA5α-pGEMT-B7-PE40KDEL, was deposited at Jun. 27, 2011 in the deposition number CGMCC NO. 4987 at China General Microbiological Culture Collection Center, No 1 Building, No. 3, Beichen west Road, Chaoyang district Beijing, China). The expression vector pcDNA3.1/Zeo(+) was stored in our department. ZeocinTM, TRIzol, and LipofectamineTM2000 were purchased from Invitrogen Corp. DMEM/F12 medium was purchased from Gibco Inc. The Jurkat and Raji cell lines were stored in our department. The CHO-K1-RPE.40 cell line was established by J M Moehring and T J Moehring and was kindly provided by Dr. Sucic Joseph. The rabbit polyclonal anti-Pseudomonas exotoxin A antibody was purchased from Sigma Inc. CD80 monoclonal antibody was purchased from R&D Inc. PVDF membrane (polyvinylidene fluoride) and Amicon Ultra-4 were purchased from Millipore Inc. Super enhanced chemiluminescence detection reagents was purchased from Pierce Inc. MTS (CellTiter 96 AQueous One Solution Cell Proliferation Assay), and the PureYieldTM plasmid midiprep system were purchased from Promega Co. Ltd. A reverse transcription kit, SYBR® Premix Ex TaqTM, Kpn I, Xba I, and T4 ligase were purchased from TaKaRa Bio. Inc. TMB (3,3′,5,5′-Tetramethylbenzidine) chromogenic substrate solution and 2×Pfu PCR MasterMix were purchased from Tiangen Biotech (Beijing) Co., Ltd. The QIAquick gel extraction kit was purchased from QIAGEN Inc.
1.2 Main Instruments
The main instruments used included a 9700 PCR instrument (PerkinElmer), Du® 640 ultraviolet detector (Beckman), Mini II protein electrophoresis instrument and protein semi-dry electrotransfer instrument (Bio-Rad), Gel-Pro3.1 gel imaging system (Media Cybernetics), 550 automatic microplate reader, and real-time quantitative PCR instrument (Stratagene, Mx3005P).
1.3 Construction of Eukaryotic Expression Vector pcDNA3.1/B7-1-PE40KDEL
The upstream primer (P1) containing the signal peptide sequence and Kpn I restriction sites and the downstream primer (P2) containing the Xba I restriction sites were designed. The signal peptide sequence was based on the sequence of the pSecTag2-B vector of Invitrogen Inc., Murine Ig k-chain V-J2-C signal peptide, which can be secreted in a large amount into the extracellular space. The sequences of the primers were as follows:
Upstream Primer P1:
5′GGTACCTGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTT CCACTGGTGACGTTATCCACGTGACCAAGGAAGTG 3′ (SEQ ID NO: 4, in which the underlined section is the Kpn I restriction site, the framed section is the start codon, and the italicized section is the signal peptide coding sequence).
Downstream Primer P2:
P2: 5′TCTAGATTACAGCTCGTCCTTCGGCGG 3′ (SEQ ID NO: 5, in which the underlined section is the Xba I restriction site)
Because the P1 sequence with the added signal peptide sequence was too long, the spliced overlap extension (SOE)-PCR method was used to split P1 into two primers for overlapping amplification.








P1-A:



(SEQ ID NO: 6)



5′ CTGCTCTGGGTTCCAGGTTCCACTGGTGACGTTATCCACGTGACCAAGGAAGTG 3′



 


P1-B:


(SEQ ID NO: 7)



5′ GGTACCTATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCA 3′







A prokaryotic expression vector, GEMT-B7-1-PE40KDEL plasmid, was used as the template, and high-fidelity Pfu DNA polymerase was used for PCR amplification. The reaction system was as follows:
















2x Pfu PCR Master Mix
12.5 μl 



P1 (20 μM)
0.5 μl



P2 (20 μM)
0.5 μl



pGEMT-B7-1-PE40KDEL (1:50)
  3 μl



ddH2O
8.5 μl



Total Volume
 25 μl










The PCR reaction conditions were as follows: pre-denaturing at 96° C. for 5 min, denaturing at 96° C. for 1 min, annealing at 63° C. for 1 min, and extension at 72° C. for 2 min for 30 cycles, followed by extension at 72° C. for 10 min. The products were resolved using 1% agarose gel electrophoresis.
The PCR products were recovered. The recovered PCR products and pcDNA3.1/Zeo(+) vector were double-digested with Kpn I+Xba I, and the digested products were recovered after electrophoresis. The digestion system was as follows:













10x Buffer M
2 μl


Xba I
1 μl


Kpn I
1 μl


0.1% BSA
2 μl


Recovered PCR Products and pcDNA3.1/Zeo(+) Vector
14 μl 


Total Volume
20 μl 









The mixture was placed in a water bath at 37° C. for 3 h.
The digested products were recovered according to the following steps:

    
    
        1) The target band from the agarose gel was cut off with a clean blade and placed in a 1.5 ml EP tube.
        2) The gel was weighed, and a corresponding volume of Buffer QG was added at the ratio of 100 mg gel: 300 μl Buffer QG.
        3) The gel was incubated in a water bath at 50° C. for 10 min until it was completely dissolved, and the tube was then inverted upside down every 2-3 minutes to mix thoroughly.
        4) One volume of isopropanol was added and mixed thoroughly.
        5) The sample was loaded into a QIAquick column and centrifuged for 1 min. After the waste liquid was discarded, 0.5 ml Buffer QG was added, and the sample was centrifuged for an additional minute.
        6) After the waste liquid was discarded and 0.75 ml of Buffer PE was added, the column rested for 2-5 min and was centrifuged for 1 min.
        7) After the waste liquid was discarded, the column was centrifuged for 1 min and then placed into a clean 1.5 ml EP tube.
        8) Thirty microliters of injection-grade H2O was added to the center of the column membrane, and after resting for 1 min, the tube was centrifuged for 1 min. The eluent was then collected.
        9) The double-digested PCR products and pcDNA3.1/Zeo(+) vector were ligated. The ligation reaction was as assembled follows:
    
    


















2x T4 Ligase Buffer
5 μl



T4 Ligase
1 μl



Double-digested PCR Products
3 μl



Double-digested pcDNA3.1/Zeo(+) Vector
1 μl



Total Volume
10 μl 










The tube filled with ligation reaction was placed in 4° C. ice water to overnight.
The following method was used to transfect the ligation product into DH5a competent bacteria:
1) Five microliters of ligation product and 20 μl of reagent A were diluted with sterile water to 100 μl; the reaction was then placed on ice for later use.
2) DH5a competent bacteria were thawed on ice (5 min), and the diluted plasmid from above was then added.
3) The mixture was placed on ice for 20 min and then rested at room temperature for 10 min. The bacteria were plated onto culture plates and incubated at 37° C. overnight.
Ampicillin-resistant bacterial clones were selected to extract plasmids, which were then screened by double-digestion with restriction enzymes (the same conditions as described above). The positive clones were sent to TaKaRa Bio., Inc. for sequencing.
The bacterial clones with the correct sequence were cryopreserved, cultured and deposited. the strain deposited in the name of Escherichia coli DHA5α-pcDNA3.1/B7-1-PE40KDEL at Jun. 27, 2011 in China General Microbiological Culture Collection Center (No 1 Building, No. 3, Beichen west Road, Chaoyang district Beijing, China), the deposition number is CGMCC NO. 4986). The PureYieldTM plasmid extraction kit was used to extract and purify large amounts of plasmids. The purified plasmids were dissolved in saline with an OD260/280 ratio of 1.8-2.0 and a concentration of >0.5 μg/μl.
1.4 Transient Expression of Recombinant Plasmid pcDNA3.1/B7-1-PE40KDEL in CHO-K1-RPE.40 cells
After 0.5×105-2×105 CHO-K1-RPE.40 cells were seeded into 6-well plates (2 ml of medium: DMEM/F12, 7.5% FBS, 1× non-essential amino acids), the Liposome transfection method was adopted: 4 μl of plasmid and 10 μl of LipofectamineTM2000 were each diluted with 250 μl of OPTI-MEM I medium and were then mixed and incubated for 20 min. The mixture was slowly added to the 6-well plates, which were then incubated at 37° C., 5% CO2 for 6 h. Then, the medium was replaced with complete DMEM medium. After 48 h, the cells and supernatant were collected and screened using RT-PCR and Western blot analysis.
1.5 RT-PCR Analysis of B7-1-PE40KDEL mRNA in Transfected Cells
Forty-eight hours after transfection, CHO-K1-RPE.40 cells in growth phase were rinsed twice with PBS. Total RNA was extracted in accordance with the instructions of the TRIzol kit and was then reverse-transcribed into cDNA for PCR amplification using the aforementioned P1 and P2 primers. β-actin was used as the reference control.
The reverse transcription reaction was assembled as follows:
















MgCl2
2 μl



10x RNA PCR Buffer
1 μl



dNTP Mixture
1 μl



RNase Inhibitor
0.25 μl  



AMV
0.5 μl  



Oligo dT
0.5 μl  



RNA
4.75 μl  



Total Volume
10 μl 










The reaction conditions were 42° C. for 30 min, 99° C. for 5 min, then 5° C. for 5 min.
The PCR reaction system was as follows:
















MgCl2
 3 μl



10x LA Buffer
 4 μl



ddH2O
31.75 μl  



LA Taq
0.25 μl  



P1 (20 μm)
0.5 μl 



P2 (20 μm)
0.5 μl 



First-Strand cDNA
10 μl



Total Volume
50 μl










The reaction conditions were as follows: pre-denaturing at 96° C. for 5 min, denaturing at 96° C. for 1 min, annealing at 55° C. for 1 min, and extension at 72° C. for 3 min for 30 cycles, followed by extension at 72° C. for 10 min. The PCR products were identified with 1% agarose gel electrophoresis.
The sequences of the β-actin primers were as follows:
Upstream primer P1: 5′CTG TGG CAT CCA CGAAAC TA 3′ (SEQ ID NO: 8)
Downstream primer P2: 5′ACA TCT GCT GGA AGG TGG AC 3′ (SEQ ID NO: 9)
1.6 Western Blot Analysis of B7-1-PE40KDEL Protein Expression in Transfected Cells
1) Protein electrophoresis: Forty-eight hours after transfection, the supernatant of the CHO-K1-RPE.40 cell culture was collected and concentrated. Subsequently, 15 μl of sample and 15 μl of 2×SDS loading buffer were combined and boiled for 5 min for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein electrophoresis was conducted at 80 V until protein bands had migrated out of the stacking gel, followed by electrophoresis at 150 V until the protein bands had reached the bottom of the separating gel, at which point the power was disconnected. The formulation of the protein electrophoresis was as follows:














10% Separating gel
5% Stacking gel



(5 ml)
(2 ml)


















ddH2O
1.90 ml
1.40 ml


30% Acrylamide
1.70 ml
0.33 ml


1.5M Tris-HCl (pH 8.8)
1.30 ml
—


1.0M Tris-HCl (pH 6.8)
—
0.25 ml


10% SDS
0.05 ml
0.02 ml


10% Ammonium persulfate
0.05 ml
0.02 ml


TEMED
0.002 ml 
0.002 ml 









2) Membrane transfer: proteins were electro-transferred to a polyvinylidene difluoride (PVDF) membrane. Immobilon-P was selected as the PVDF membrane, which was soaked in methanol for 15 s, then in water for 2 min, and then in electro-transfer buffer for 20 min. Meanwhile, the filter paper and gel were soaked in electro-transfer buffer for 15 min. The transfer system was set up as +(white)/three layers of filter paper/membrane/gel/three layers of filter paper/black. The transfer was performed at 60 mA for 40 min.
3) The membrane was blocked at room temperature for 2 h.
4) Primary antibody, rabbit anti-PEA antiserum, or CD80 monoclonal antibody that was appropriately diluted with the blocking solution was added, and the membrane was incubated at 4° C. overnight.
5) The membrane was washed with TBST 3 times for 5 min each time.
6) HRP-anti-rabbit or HRP-anti-goat IgG secondary antibody diluted with the blocking solution was added, and the membrane was incubated at room temperature for 1 h.
7) The membrane was washed with TBST 3 times for 10 min each time.
8) The ECL method was used to develop images of the gels.
1.7 Stable Transfection of CHO-K1-RPE.40 Cells with Recombinant Plasmid pcDNA3.1/B7-1-PE40KDEL
Liposome transfection was performed with the same procedures outlined in section 1.4. Twenty-four hours after transfection, the transfected cells were diluted at a 1:20 ratio, and ZeocinTM was added to a concentration of 800 g/ml. The medium was replaced every 4 days, and cells were seeded into 24-well plates as the Zeo (+) clones gradually increased in size. Once the cells reached confluence, they were seeded into 6-well plates for continual culturing in multiple plates. Cells were identified by the real-time quantitative RT-PCR method, and positive cell clones were cryopreserved and stored for later use.
1.8 Enzyme-Linked Immunosorbent Assay (ELISA) Analysis of B7-1-PE40KDEL Protein Expression in Transfected Cells
1) Forty-eight hours after transfection, the supernatant of the stably transfected cell culture was collected and concentrated.
2) Ten microliters of the concentrate was diluted at a 1:10 ratio and was then added to 96-well plates overnight. The volume in each well was 1001, and three parallel wells were set up for each sample.
3) The samples were blocked with blocking solution for 1 h.
4) Anti-PEA polyclonal antibody was added and incubated at 37° C. for 1 h.
5) The plates were rinsed with PBST 3 times.
6) HRP-anti-rabbit IgG was added and incubated at 37° C. for 1 h.
7) The plates were rinsed with PBST 3 times.
8) Chromogenic TMB solution was added, and 15 min later, the reaction was terminated with 2 mol/L H2SO4. The absorbance values were measured at 450 nm, and the corresponding concentrations were calculated.
9) The untransfected cells were used as a blank control, and PEA was used as the standard to graph the standard curve (Kirman J R, Seder R A. DNA vaccination: the answer to stable, protective T-cell memory? Curr Opin Immunol, 2003, 15: 471-476).
1.9 Selective Cytotoxicity Analysis of Eukaryotic Expressed B7-1-PE40KDEL
Human lymphoma cell line Jurkat cells with a high expression level of CD28 and positive CHO-K1-RPE.40 cells stably transfected with pcDNA3.1/B7-1-PE40KDEL were co-cultured in 96-well plates at a concentration of 1×105/ml at 37° C. and 5% CO2 for 48 h. Then, 20 μl of MTS was added; 1 h later, the absorbance values at 490 nm were measured, and cytotoxic activity was calculated. The human Burkitt's lymphoma cell line Raji with a lower expression level of CD28 was used as a negative control. The cell death rate was calculated as follows:

Cell death rate=(1−the number of surviving cells in the well of co-cultured stably transfected CHO-K1-RPE.40 cells and target cells/the number of surviving cells in the well of co-cultured untransfected CHO-K1-RPE.40 cells and target cells)×100%.

2. Results
2.1 Construction of Eukaryotic Expression Vector pcDNA3.1/B7-1-PE40KDEL
To express B7-1-PE40KDEL fusion gene in eukaryotic cells, a eukaryotic expression vector pcDNA3.1/B7-1-PE40KDEL which contains B7-1-PE40KDEL and the Zeo (+) gene, was constructed as shown in FIG. 1. The designed PCR primers were used to amplify human B7-1-PE40KDEL, which was analyzed by agarose gel electrophoresis. The expected specific band of 1,850 bp was observed (FIG. 2). The recovered double-digested products were cloned into the Kpn I and Xba I restriction sites of the eukaryotic expression vector pcDNA3.1/Zeo(+) to construct the recombinant plasmid pcDNA3.1/B7-1-PE40KDEL, which was then extracted, electrophoresed (FIG. 3), and screened by sequencing (FIG. 4. The sequence is shown in Appendix 1) to obtain positive clones. The sequencing results revealed no point mutations or frameshift mutations in the sequence following the signal peptide sequence and were consistent with the sequence of the prokaryotic expression plasmid pRSETA-B7-1-PE40KDEL. The comparison results of the primary and secondary protein structures between B7-1-PE40KDEL, human B7-1, and PE40 are shown in Table 1.







TABLE 1







Comparison of the primary and secondary protein structures between


B7-1-PE40KDEL fusion protein, B7-1, and PE40.









B7-1-PE40 KDEL











Secondary


Structure
Primary structure
structure





B7-1-208 (1-208aa)
 25aa Leucine→Proline
No change



156aa Histidine→Arginine


Link-19 (209-227aa)


PE40-360 (228-587aa)
247aa Phenylalanine→Serine
No change



397aa Valine→Isoleucine
No change









2.2 Expression Analysis of B7-1-PE40KDEL Exotoxin Fusion Gene from Eukaryotic Cells
The eukaryotic expression vector pcDNA3.1/B7-1-PE40KDEL with the correct sequence was transiently transfected into CHO-K1-RP E.40 cells, and the following methods were used to detect the expression of B7-1-PE40KDEL fusion protein: first, RT-PCR was used to detect the expression of B7-1-PE40KDEL mRNA in the transfected cells. A specific band of 1,850 bp was present in CHO-K1-RPE.40 cells transfected with pcDNA3.1/B7-1-PE40KDEL, whereas the specific band was not observed in CHO-K1-RPE.40 cells transfected with an empty vector. β-actin PCR products were used as an internal reference (FIG. 4). Western blot analysis was then conducted to detect the antigenicity and secretion expression of the B7-1-PE40KDEL fusion protein in the cell culture supernatant. The results revealed a positive band of 62-83 kDa using either a rabbit anti-PEA antiserum or a CD80 monoclonal antibody, whereas no band was observed from cells transfected with an empty vector (FIG. 5).
2.3 Screening of Stably Transfected Cells and Expression Analysis of B7-1-PE40KDEL Fusion Protein
Real-time semi-quantitative analysis of the screened out 40 clones indicated that 16 clones were positive. Those positive clones were then cultured and cryopreserved in batch (FIG. 6). To detect the amount of secretion expression of B7-1-PE40KDEL in stably transfected cells, the cell culture supernatants from a number of positive clones were sampled to graph the standard curve of protein concentration using ELISA and the known concentrations PEA as standards (Table 2, FIG. 7). The OD value of each supernatant from these samples was compared to the standard curve to obtain the secreted amount in each stably transfected clone. The results are presented in Table 3. The results indicated that 24 h post-transfection, approximately 287 pg/ml of PEA protein was expressed in 1×106 stably transfected cells.







TABLE 2







ELISA detection values of different concentrations


PEA standard substance










PEA Concentration (pg/ml)
OD Value














0
0.105



78.125
0.177



156.25
0.232



312.5
0.47



625
0.627



1250
1.143



2500
1.772



5000
2.477










OD450 was used as the ordinate and PEA concentrations as the abscissa to graph the standard curve using Curve Expert 1.3 software. The standard curve of the polynomial fitting equation (FIG. 7) was used to calculate the PEA concentrations from the culture supernatant of stably transfected cell strains.







TABLE 3







The expression levels of B7-1-PE40KDEL fusion proteins


in different positive clones













PEA Concentrations



Clone No.
OD450 Value
(pg/ml)















Clone 1*
0.352
268.26



Clone 2*
0.317
230.3



Clone 4
0.385
303.98



Clone 32
0.425
347.28










2.4 Selective Cytotoxicity of the Eukaryotic Expressed B7-1-PE40KDEL
The human lymphoma cell line Jurkat with a high expression level of CD28 was used to detect the selective cytotoxic activity of the eukaryotic expressed B7-1-PE40KDEL. The human Burkitt's lymphoma cell line Raji with a low expression level of CD28 was used as a negative control. The MTT results indicated that after 48 h of co-incubation of stably transfected cells and Jurkat cells, the OD value was 0.782, whereas the OD values were 1.466 for untransfected cells, 1.29 for Jurkat cells alone, 1.296 for the co-incubation of stably transfected cells and Raji cells, and 1.564 for untransfected cells alone. According to the equation, the The percentage of cell killing of Jurkat cells and Raji cells by the stably transfected cells were 92.87% and 15%, respectively, these result confirmed that the eukaryotic expressed B7-1-PE40KDEL have a good selective inhibitory effect on CD28-positive cells in vitro. (FIG. 8).
Example 2
The Protection Against aGVHD by B7-1-PE40KDEL DNA Vaccine (1)
1. Materials and Methods
1.1 Plasmids, Cells, and Main Reagents
The eukaryotic expression vector pcDNA3.1/B7-1-PE40KDEL was constructed by our group. This eukaryotic expression vector was previously constructed and stored by Dr. Hong Xue. The rabbit polyclonal anti-Pseudomonas exotoxin A antibody was purchased from Sigma Inc. FITC-anti-mouse CD3, PE-anti-mouse CD28, and PharmlyseTM were purchased from BD Inc. SYBR® Premix Ex TaqTM (perfect real-time) was purchased from TaKaRa Bio. Inc. MTX for injection was purchased from Jiangsu Hengrui Medicine Co., Ltd., and cyclosporin A was purchased from Novartis Pharma Schweiz AG. Mouse lymphocyte separation solution was purchased from Tianjin Hao Yang Biological Products Co. Ltd.
The donor male mice C57BL/6 (H2b) and The recipient female mice Balb/c (H2d) housed in specific pathogen-free conditions weighed 16-18 g and 18-22 g at the beginning of the experiments. All experimental animals were provided by the Animal Center of the Academy of Military Medical Sciences, and the breeding conditions were at the specific pathogen-free (SPF) level. The Department of Pathology at the Affiliated Hospital of the Academy of Military Medical Sciences assisted in the preparation and observation of pathological samples. Flow cytometric analysis was conducted by the Institute of Radiation Medicine of the Academy of Military Medical Sciences. The real-time quantitative PCR analysis was conducted by the blood center of the Affiliated Hospital of the Academy of Military Medical Sciences.
1.2 Main Instruments
A WJ2002 in vivo gene transfer instrument (Scientz Biotechnology Co. Ltd.) and a 550 automatic microplate reader (Bio-Rad Laboratories, Inc.) were used in our study.
1.3 Establishment of an aGVHD Mouse Model
The recipient mice were provided with drinking water that contained gentamicin (32×104 U/L) and erythromycin (250 mg/L) one week prior to the transplantation to prevent infection and were fed in a sterile laminar airflow cabinet. 60Co total body irradiation (TBI) (8.0 Gy, dose rate is 1.8 Gy/min) was administered 4 h prior to transplantation.
The preparation of donor bone marrow cells and spleen cells was as follows: C57BL/6 donor mice were sacrificed by cervical dislocation and then soaked in 75% ethanol for a few minutes. The femur was sterilely obtained with surgical scissors, and RPMI-1640 culture medium was used to flush out the bone marrow from the bone marrow cavity, which was then sifted through a 200-mesh cell sieve. Next, 0.83% ammonium chloride solution was used to lyse the erythrocytes, followed by two washes with RPMI-1640 culture medium (1000 rpm, 10 min). This produced a single-cell suspension of bone marrow cells. The cell concentration was then adjusted to 1×108/ml. In addition, the spleens were removed aseptically and then ground on a 200-mesh cell sieve. After the cells were collected by centrifugation, 0.83% NH4Cl solution was used to lyse the erythrocytes, followed by two washes with RPMI-1640 culture medium (1000 rpm, 10 min). The resulting single-cell suspension of spleen cells was then adjusted to a cell concentration of 1×108/ml.
The prepared spleen cells and bone marrow cells were infused via tail vein injections into the recipient mice of all groups. The numbers of infused cells were 2×107 spleen cells/mouse and 1×10 bone marrow cells/mouse. The establishment of a stable aGVHD mouse model was confirmed with physical signs, the assessment of hematopoietic reconstruction, pathological analysis, and chimerism analysis after transplantation.
1.4 Protection Protocols Against aGVHD by B7-1-PE40KDEL DNA Vaccine in aGVHD Mice
Sixty aGVHD mice were randomly divided into six groups with 10 mice in each group: (1) B7-1-PE40KDEL DNA vaccine group; (2) B7-2-PE40KDEL DNA vaccine group; (3) B7-1-PE40KDEL+B7-2-PE40KDEL DNA vaccine group; (4) empty vector group; (5) CsA+MTX positive control group; and (6) untreated aGVHD group. Both the empty vector and DNA vaccine vector were dissolved in normal saline. There was no RNA contamination of the plasmid vector. Endotoxin was removed. Supercoiled DNA accounted for 70%-80% of the DNA. The OD260/OD280 ratios ranged from 1.8-2.0. The concentration was not less than 0.5 μg/μl. The preliminary experiment indicated that when a 75 g of B7-1-PE40KDEL DNA vaccine was infused, the protection against aGVHD was good with few side effects. Immediately after transplantation, 75 μg of empty vector or DNA vaccine vector was injected into mice quadriceps, and electrical impulse-mediated stimulation was given at the injection site to efficiently deliver the genes into the body. The pulse parameters were as follows: voltage, 200 V/cm; pulse width, 10 ms; the number of pulses, 6; frequency, 1 Hz (Mir L M, Bureau M F, Gehl J, et al. High-efficiency gene transfer into skeletal muscle mediated by electric pulses. Proc. Natl. Acad. Sci. USA, 1999, 96: 4262-4267). The “gold standard” regimen CsA+MTX that has been commonly used in clinical practice, was adopted as the control drugs. CsA was administered via intraperitoneal injection once per day at a dose of 1.5 mg/kg·d. MTX was administered via intraperitoneal injection on days 1, 3, 6, and 11 at a dose of 0.4 mg/kg·d (Xu K, Li C, Pan X, et al. Study of Relieving Graft-versus-Host Disease by Blocking CD137-CD137 Ligand Costimulatory Pathway in Vitro. Int J Hematol, 2007, 86:84-90.).
1.5 Expression of B7-1-PE40KDEL DNA Vaccine in Normal Mice
Electrical impulse-mediated intramuscular injection was used to inject 75 μg of purified B7-1-PE40KDEL DNA vaccine into the quadriceps of normal Balb/C mice, followed by electrical stimulation (200 V/cm, 10 ms, 1 Hz, 6 pulses). On days 1, 7, 14, 21, 28, and 35 after injection, the eyeballs were removed to collect blood, which was then treated with heparin. A TRIzol kit was used to extract RNA, and RT-PCR was used to qualitatively detect B7-1-PE40KDEL mRNA expression. Real-time quantitative PCR was further used to compare the blood expression levels of PE40 mRNA. β-actin was used as an internal reference.
RNA extraction was performed as follows: mouse lymphocyte-separating solution was used to isolate white blood cells from the peripheral blood of mice (approximately 1 ml), which were washed twice with PBS before 1 ml of TRIzol was added. The isolated cells rested at room temperature for 5 min, and 200 μl of chloroform was then added and mixed well by shaking. After stratification, the mixture was centrifuged at 4° C. and 12,000 g for 15 min. The upper aqueous phase was transferred to another tube, and 500 μl of isopropanol was added and mixed well. After resting at room temperature for 10 min, the mixture was centrifuged at 4° C. and 12,000 g for 10 min. The supernatant was discarded, and RNA was at the bottom of the tube, which was washed with 75% ethanol and then centrifuged at 4° C. and 8000 g for 5 min. The supernatant was then discarded. After the complete evaporation of ethanol, RNA was dissolved with an appropriate amount of DEPC-treated water and quantitatively measured with a UV spectrophotometer.
The reaction conditions of reverse transcription were as follows:

















MgCl2
2
μl



10x RNA PCR Buffer
1
μl



dNTP
1
μl



RNase Inhibitor
0.25
μl



AMV
0.5
μl



Oligo dT
0.5
μl



RNA
0.5
μg



Total volume
10
μl










The reaction conditions were 42° C. for 30 min, 99° C. for 5 min, and 5° C. for 5 min.
The sequences of mouse β-actin primers were:
Upstream primer P1: 5′AGG CAT CCT GAC CCT GAA GTA C 3′ (SEQ ID NO: 10)
Downstream primer P2: 5′TCT TCA TGA GGTAGT CTG TCA G 3′ (SEQ ID NO: 11)
The PCR reaction was assembled as follows:
















SYBR ® Premix Ex Taq TM (2x)
12.5 μl 



ddH2O
10.5 μl 



P1 (20 μM)
0.5 μl



P2 (20 μM)
0.5 μl



cDNA
  1 μl



Total Volume
 25 μl










Real-time PCR reaction:
Segment 1: pre-denaturing
Repeat: 1 cycle
95° C. for 5 min
Segment 2: PCR reaction
Repeat: 35 cycles
94° C. for 30 s
59° C. for 30 s
72° C. for 60 s
Segment 3: Dissociation.
1.6 In Vivo Activity of B7-1-PE40KDEL DNA Vaccine in Normal Mice
On days 1, 7, 14, 21, 28, and 35 after the injection of 75 μg of purified B7-1-PE40KDEL DNA vaccine, the mice eyeballs were removed, and blood was collected and treated with heparin. One microliter of both FITC-anti-mouse CD3 and PE-anti-mouse CD28 was added to 100 μl of anticoagulant blood, followed by incubation at room temperature in the dark for 30 min. Next, 2 ml of PharmlyseTM erythrocyte lysis buffer was added, followed by incubation at room temperature in the dark for 15 min. The mixture was then centrifuged at 1500 rpm for 5 min. After the supernatant was discarded, the pellet was re-suspended with PBS followed by centrifugation at 1500 rpm for 5 min. After the supernatant was discarded, 0.5 ml of 2% paraformaldehyde-PBS was added for detection.
1.7 The Detection of Anti-PEA Antibody in Normal Mice Treated with B7-1-PE40KDEL DNA Vaccine (Chen S Y, Yang A G, Chen J D, et al. Potent Antitumor Activity of a New Class of Tumor-Specific Killer Cells. Nature, 1997, 385:78-80.)
On day 21 after the intramuscular injection of B7-1-PE40KDEL DNA vaccine, the mice eyeballs were removed, the blood was collected and centrifuged, and the serum was stored at −20° C. for later use. ELISA was performed as follows: PEA (1 mg/mL) was dissolved in coating buffer, and each well was coated with 200 ng/1001, followed by incubation at 4° C. overnight. The next day, the wells were washed 2-3 times with PBST and then blocked in bovine serum blocking solution at room temperature for 2 h. The serum sample and standard samples (100 μl/well) were incubated at 37° C. for 1 h and then washed 3 times with PBST. Next, 100 μl of HRP-anti-mouse IgG diluted at a ratio of 1:50,000 was added to each well and incubated at 37° C. for 1 h. After washing 4 times with PBST, 100 μl of TMB chromogenic solution was added to each well and incubated at 37° C. for 15 min. The reaction was terminated by adding 50 Cl of 2 M H2SO4 to each well, and the absorbance values were measured at 450 nm. The rabbit anti-PEA polyclonal antibody was used as a positive control, and the serum from mice injected with pcDNA3.1 empty vector was used as a negative control.
1.8 The Toxicity and Side Effects of B7-1-PE40KDEL DNA Vaccine
After the intramuscular injection of 75 g of B7-1-PE40KDEL DNA vaccine, the physical signs and behavior of the mice were observed. In addition, on days 7, 14, and 21, the heart, liver, spleen, lung, and kidney were taken for histopathological examination to make preliminary observations as to whether there was toxicity or side effects of B7-1-PE40KDEL DNA vaccine in mice.
1.9 The Effect of B7-1-PE40KDEL DNA Vaccine on the Physical Signs of GVHD Mice
The mice in all groups were weighed each day, and physical signs such as hair, spirit, and diarrhea were observed.
1.10 The Effect of B7-1-PE40KDEL DNA Vaccine on Hematopoietic Reconstruction in GVHD Mice
After the preconditioning, 20 μl of blood was collected from the tail vein and diluted with 3801 of white cell diluent for white blood cell counts with hemacytometer at regular intervals, which was used to understand the changes in blood and hematopoietic reconstruction in mice from all groups.
1.11 The Effect of B7-1-PE40KDEL DNA Vaccine on Chimerism in GVHD Mice
Different genders of mice were used as the donor and recipient in bone marrow transplantation.
Therefore, the genomic DNA could be extracted from the peripheral blood of female recipient mice and the Y chromosome-specific gene sry primers could be designed to PCR-amplify the Y chromosome of male donor mice to detect transplant engraftment.









sry primer 1:



(SEQ ID NO: 12)



5′-TGTGGTCCCGTGGTGAGA-3′;



 



primer 2:



(SEQ ID NO: 13)



5′-ATCAACAGGCTGCCA ATAAA-3′.






1.12 The Effect of B7-1-PE40KDEL DNA Vaccine on Histopathology in GVHD Mice
On day 21 after transplantation, the livers, spleens, small intestines, and skin of mice in all groups were removed and fixed in 10% formalin solution, followed by paraffin sectioning and HE staining to observe the pathological changes.
1.13 Statistical Analysis
SPSS 13.0 software was used for statistical analysis. The t tests were used to test the statistical significance of the differences in the mean measurement data between the two groups. A non-parametric test (the Mann-Whitney test) was used to compare ranked data between the two groups. P<0.05 was considered statistically significant. SPSS 13.0 software was also used to graph the Kaplan-Meier survival curve.
2. Results
2.1 The Expression of B7-1-PE40KDEL DNA Vaccine in Normal Mice
The RT-PCR results (FIG. 9) indicated that on days 1, 7, 14, 21, 28, and 35 after the injection of the B7-1-PE40KDEL DNA vaccine, B7-1-PE40KDEL mRNA was detectable. We used 3-actin as an internal reference.
Real-time quantitative PCR (FIG. 10) showed that the expression of PE40KDEL mRNA was relatively higher on days 1 and 7, and then the expression level gradually declined with time to the lowest levels on days 28 and 35.
2.2 The In Vivo Activity of B7-1-PE40KDEL DNA Vaccine in Normal Mice
Because B7-1-PE40KDEL can specifically kill cells with high expression levels of CD28, flow cytometric analysis was conducted to analyze the removal of CD28+T cells from the peripheral blood of mice injected with B7-1-PE40KDEL DNA vaccine, which was indicated as geometric mean fluorescence intensity (Geo mean) (FIG. 11). The results showed that on day 7 after the intramuscular injection of B7-1-PE40KDEL DNA vaccine, the effect of CD28+T removal began to appear, and the Geo mean of blood CD28+T cells was lower than that of normal control mice. On day 21 after injection, CD28+T count declined to the lowest level. On days 28 and 35 after injection, CD28+T count gradually recovered, but the Geo mean of CD28+T cells was still lower than that of normal mice. The results suggested that with the declining expression level of B7-1-PE40KDEL, CD3+CD28+T cells showed a trend towards recovering to a normal level (FIG. 12).
2.3 Detection of Serum Anti-PEA Antibody Levels in Mice after the Intramuscular Injection of B7-1-PE40KDEL DNA Vaccine
The anti-PEA antibody level was detected using ELISA. A commercial anti-PEA antibody was used as the standard to graph the standard curve. The OD450 was used for the abscissa, and anti-PEA antibody concentration was used for the ordinate to graph the standard curve using CurveExpert 1.3 software (FIG. 13). A quadratic fitting equation was used to calculate concentrations of serum anti-PEA antibodies (Table 4). The anti-PEA antibody level of B7-1-PE40KDEL DNA vaccine intramuscular-injection group was significantly lower than that of the positive control group (p<0.05), and there was no significant difference when compared to that of the negative control group (i.e., the empty-vector injection group).







TABLE 4







Serum anti-PEA antibody levels in mice on day 21 after the intramuscular


injection of B7-1-PE40KDEL DNA vaccine










Absorbance Value
Concentration


Groups
(450 nm)
(pg/ml)





pcDNA3.1/B7-1-PE40KDEL
0.283 ± 0.029*
 63.824 ± 6.402*


(75 μg)


pcDNA3.1 group
0.139 ± 0.022*
 49.814 ± 1.669*


(negative control)


Positive control
0.564 ± 0.084
312.458 ± 3.842





*indicates the comparison with the positive control, P < 0.05






2.4 Toxicity and Side Effects of B7-1-PE40KDEL DNA Vaccine in Normal Mice
After the intramuscular injection of 75 g of B7-1-PE40KDEL DNA vaccine, the mice were observed every day, and no abnormalities in physical signs or behavior were observed. Histopathological examination of hearts, livers, spleens, lungs, and kidneys removed on days 7, 14, and 21 after the injection did not reveal any pathological changes.
2.5 Establishment of an aGVHD Mouse Model
One week after transplantation, recipient mice began to experience typical GVHD symptoms, such as changes in hair color, listlessness, loss of appetite, serious epilation in the abdomen and head, diarrhea, black stool, ulcers, and hunched posture (FIG. 14). Their average body weight progressively declined at nearly 1 g/day and reached a low point of approximately 14.77±1.66 g on day 10. Afterwards, body weight began to increase and then began to gradually decline again after day 14 through death. The total weight loss was approximately 8 g at death (FIG. 15). White blood cell counts in the peripheral blood began to drop on day 1 and were lowest (0.69±0.18×109/L) on day 3 ̃day 4. The count began to recover on day 6 and reached a peak on day 14, then continued to drop afterwards. The white blood cell count was 1.54±0.14×109/L at death. On day 35 after transplantation, the specific Y-chromosome sry gene sequence of male donor mice could be detected in the peripheral blood of all recipient mice, as shown in FIG. 16 as the 371-bp fragment, suggesting that the transplantation was successful. Pathological examination revealed typical GVHD pathological findings as follows: {circle around (1)} Necrosis of the glandular epithelial cells in the intestinal mucosa was observed in small intestine tissue, and necrotic cell debris was present in the epithelial cells and glandular cavity; glandular epithelial cells were flat, glands were cystic, the number of glands was reduced, glands disappeared, and the mucosal epithelium fell off (FIG. 17-A); {circle around (2)} In liver tissue, focal liver cell degeneration and necrosis occurred, and lymphocyte and eosinophil infiltration was observed (FIG. 17-B). Liver cells swelled and degenerated; {circle around (3)} In the spleen, we observed massive bleeding, splenic fibrosis, spleen sinus expansion and bleeding, and sparse spleen cells (FIG. 17-C); {circle around (4)} Fibrous tissue hyperplasia was observed in the subdermal region, and lymphocyte infiltration occurred in the dermis (FIG. 17-D). All mice in the GVHD model group died within 24 days after radiation, with a median survival time of 22.7 days. The average survival time of the bone marrow transplantation group was more than 60 days, which can be considered long-term survival. The median survival time of the spleen cell infusion group was 9.6 days. The median survival time of radiation group alone was 12.3 days. The survival rates of the mice in all groups were depicted in a Kaplan-Meier survival curve (FIG. 18). In the summation of the typical GVHD symptoms listed above, we confirmed that a stable, acute GVHD mouse model had been established, and the success rate was nearly 100%.
2.6 The effects of B7-1-PE40KDEL and B7-2-PE40KDEL DNA vaccines on the physical signs of aGVHD mice After the treatment with B7-1-PE40KDEL DNA vaccine (called as B7-1), B7-2-PE40KDEL DNA vaccine (called as B7-2), and B7-1-PE40KDEL+B7-2-PE40KDEL DNA vaccine (called as B7-1+B7-2), the symptoms of hunched posture and epilation were significantly improved compared with the other groups. The starting time of epilation in the combination treatment group was 3-6 days later than in other groups. There was no blood in the stool and no perianal swelling. The weight loss of the mice was the smallest in B7-1-PE40KDEL DNA vaccine group, followed by CsA+MTX group, B7-1-PE40KDEL+B7-2-PE40 KDEL DNA vaccine group, and B7-2-PE40KDEL DNA vaccine group. The mice in the empty vector group and the untreated GVHD group (NS group) experienced the greatest weight loss. The changes in body weight of GVHD mice in all groups after treatment are shown in FIG. 19.
2.7 The Effect of B7-1-PE40KDEL and B7-2-PE40KDEL DNA Vaccines on Hematopoietic Reconstruction of aGVHD Mice
The WBC counts in the peripheral blood of the aGVHD mice in all groups began to drop on day 1 to a low point of approximately 0.6×109/L on day 3 ̃day 4. The WBC count began to recover on day 6, and the level of recovery was higher in B7-1-PE40KDEL DNA vaccine, B7-2-PE40KDEL DNA vaccine, B7-1-PE40KDEL+B7-2-PE40KDEL DNA vaccine, and CsA+MTX groups than in the empty vector group and the untreated group. Of all groups, the level of WBC recovery was the highest in B7-1-PE40KDEL DNA vaccine group. Subsequently, WBC counts continued to drop again to >1.0×109/L at death in all groups (FIG. 20).
2.8 The Effect of B7-1-PE40KDEL and B7-2-PE40KDEL DNA Vaccines on Histopathology of aGVHD Mice
Pathological analysis was performed on small intestine tissues of the mice in all groups on day 21 after treatment (FIG. 21). The results revealed good glandular epithelial cell integrity and relatively few necrotic cells in group C (B7-1-PE40KDEL+B7-2-PE40KDEL DNA vaccine) and group E (CsA+MTX). In group F (untreated aGVHD group), there was massive necrosis of the mucous glandular epithelial cells, and the shedding of mucosal epithelium was the most serious. The pathological analysis of skin (FIG. 22) revealed serious fibrous tissue hyperplasia in the subdermal region in group D (empty vector group) and group F (untreated aGVHD group). In group A (B7-1-PE40KDEL DNA vaccine), group B (B7-2-PE40KDEL DNA vaccine), group C (B7-1-PE40KDEL+B7-2-PE40KDEL DNA vaccine), and group E (CsA+MTX), the lesions were less severe, and the degree of lymphocyte infiltration was also lower than in groups D and F. Pathological analysis of the liver (FIG. 23) revealed that in group A only, the degree of liver cells' swelling and hydropic degeneration was relatively low. In other groups, there was focal liver cell degeneration and necrosis, infiltration of lymphocytes and eosinophils, and more serious liver cell swelling and degeneration.
2.9 The Effect of B7-1-PE40KDEL and B7-2-PE40KDEL DNA Vaccines on the Survival Time of aGVHD Mice
The survival time of the mice in B7-1-PE40KDEL DNA vaccine group was the longest, with a median survival time of 51 days. The median survival times of B7-2-PE40KDEL DNA vaccine and B7-1-PE40KDEL+B7-2-PE40KDEL DNA vaccine groups, empty vector group, CsA+MTX group, and the untreated group were 41, 47, 22.5, 39.7, and 21.5 days, respectively. The survival rates of the mice in all groups are depicted in a Kaplan-Meier survival curve (FIG. 24).
2.10 Chimerism Evaluation of aGVHD Mice in all Groups after Treatment with B7-1-PE40KDEL and B7-2-PE40KDEL DNA Vaccines
On day 21 after transplantation, the specific Y-chromosome sry gene of the male donor mice was detectable in the peripheral blood of the recipient mice in all treatment groups (FIG. 25), suggesting that the transplantation was successful in all groups.
Example 3
The Protection Against aGVHD by B7-1-PE40KDEL DNA Vaccine (2)
1. Materials and Methods
1.1 Experimental Animals
The donor male mice C57BL/6 (H2b) and The recipient female mice Balb/c (H2d) housed in specific pathogen-free conditions weighed 16-18 g and 18-22 g at the beginning of the experiments. All experimental animals were provided by the Animal Center of the Academy of Military Medical Sciences, and the breeding conditions were at the specific pathogen-free (SPF) level.
1.2 Drugs and Reagents
The eukaryotic expression vector pcDNA3.1/Zeo(+)-B7-2-PE40KDEL was constructed and stored in our department. The eukaryotic expression vector was constructed and stored as described in section 1. MTX for injection was purchased from Jiangsu Hengrui Medicine Co., Ltd. Cyclosporin A was purchased from Novartis Pharma Schweiz AG. FITC-anti-mouse CD3, PE-anti-mouse CD28, PE-Cy5-anti-mouse CD8, PE-Cy5-anti-mouse CD4, PE-anti-mouse CD4, and PE-anti-mouse CD25 were all purchased from BD Inc. Fluorokine MAP mouse interferon (IFN) γ, interleukin (IL)-2, IL-4, IL-10, IL-12, tumor necrosis factor (TNF) α kits, and Luminex liquid chips for cytokine detection were provided by R&D Inc. Tumor growth factor (TGF) β and IL-2 ELISA kits were provided by R&D Inc.
1.3 Establishment of an aGVHD Mouse Model
The recipient mice were provided drinking water that contained gentamicin (32×104 U/L) and erythromycin (250 mg/L) one week prior to transplantation to prevent infection and were fed in a sterile laminar airflow cabinet. 60Co total body irradiation (TBI) (8.0 Gy, dose rate is 1.8 Gy/min) was administered 4 h prior to transplantation.
The preparation of donor bone marrow cells and spleen cells was as follows: C57BL/6 donor mice were sacrificed by cervical dislocation and then soaked in 75% ethanol for a few minutes. The femur was sterilely obtained with surgical scissors, and RPMI-1640 culture medium was used to flush out the bone marrow from the bone marrow cavity, which was then sifted through a 200-mesh cell sieve. Next, 0.83% NH4Cl solution was used to lyse the erythrocytes, followed by two washes with RPMI-1640 culture medium (1000 rpm, 10 min). As a result, a single-cell suspension of bone marrow cells was obtained, and the cell concentration was then adjusted to 1×108/ml. In addition, the spleens were removed aseptically and ground on a 200-mesh cell sieve. After the cells were collected by centrifugation, 0.83% NH4Cl solution was used to lyse the erythrocytes, followed by two washes with RPMI-1640 culture medium (1000 rpm, 10 min). As a result, a single-cell suspension of spleen cells was obtained, and then the cell concentration was adjusted to 1×108/ml.
The prepared spleen cells and bone marrow cells were infused via tail vein injection into the recipient mice of all groups. The numbers of infused cells were 2×107 spleen cells/mouse and 1×10 bone marrow cells/mouse. The establishment of a stable aGVHD mouse model was confirmed with the detection of physical signs, hematopoietic reconstruction, transplantation, and pathological analysis.
1.4 Protection Protocols Against aGVHD by B7-1-PE40KDEL DNA Vaccines in aGVHD Mice
The protection protocol of B7-PE40KDEL DNA vaccine and other treatment was as follows: drugs were administered to the mice of all groups at day 1 after transplantation. There were six groups with five mice in each group, as follows: (1) B7-1-PE40KDEL DNA vaccine group; (2) B7-2-PE40KDEL DNA vaccine group; (3) B7-1-PE40KDEL+B7-2-PE40KDEL DNA vaccine group; (4) empty vector group; (5) CsA+MTX control group; (6) the untreated aGVHD group; and (7) the normal control group. Both the empty vector and DNA vaccine vector were dissolved in normal saline. There was no RNA contamination of the plasmid vector. Endotoxin was removed. Supercoiled DNA accounted for 70%-80% of DNA, and the OD260/OD280 ratio ranged from 1.8-2.0. The concentration was not less than 0.5 μg/μl. Immediately after transplantation, 75 μg of empty vector or DNA vaccine vector was injected into mice quadriceps, and electrical stimulation was applied at the injection site. The pulse parameters were as follows: voltage, 200 V/cm; wave width, 10 ms; the number of pulses, 6; frequency, 1 Hz. For control drugs, the “gold standard” regimen CsA+MTX that was commonly used in clinical practice was used. CsA was administered via intraperitoneal injection once a day at a dose of 1.5 mg/kg·d, whereas MTX was administered via intraperitoneal injection on days 1, 3, 6, and 11 at a dose of 0.4 mg/kg·d.
1.5 Detection of Serum Cytokines in aGVHD Mice by Luminex Liquid Chip
On days 7, 14, and 21 after transplantation, blood was drawn from the infraorbital venous plexus of the mice. After the serum was separated with a centrifuge, the following procedures were performed. Each sample was examined in replicate.
1) Preparation before the experiment included the following:
Twenty milliliters of wash buffer concentrate was diluted to 500 ml of wash buffer;
The sample was diluted 4-fold: 37.5 μl of RD6-40+12.5 μl of sample;
Dilution of standard samples: the standard sample was diluted with 0.9 ml of RD6-40 into a standard cocktail, which was then progressively diluted into varying concentrations;
Dilution of microsphere: 50 μl of microsphere concentrate→5 ml of microsphere diluent;
Dilution of biotin-antibody: 50 μl of biotin-antibody concentrate→5 ml of biotin-antibody diluent;
Dilution of streptavidin-PE: 55 μl of streptavidin-PE concentrate→5.5 ml wash buffer.
2) All reagents, standards, and samples were prepared at room temperature.
3) One hundred microliters of wash buffer was used to pre-wet the membrane on the microplate and remove the liquid with vacuum washer.
4) The diluted microspheres were re-suspended by gently shaking the shaker, and then 50 μl of microspheres was added to each well.
5) After 50 μl of sample or standard sample was added to each well, the wells were sealed, followed by incubation at room temperature in the dark (covered with aluminum foil) for 3 h.
6) The liquid was removed with a vacuum washer, and the plate was washed 3 times with 100 μl of wash buffer.
7) After 50 μl of a diluted mixture of biotin-antibodies was added, the wells were sealed with a membrane, followed by incubation while shaking at 500±50 rpm at room temperature in the dark (covered with aluminum foil) for 1 h.
8) The plate was washed 3 times, as described in 6).
9) After 50 μl of diluted streptavidin-PE was added to each well, the wells were sealed with membrane, followed by incubation while shaking at 500±50 rpm at room temperature in the dark (covered with aluminum foil) for 0.5 h.
10) The plate was washed 3 times, as described in 6).
11) After 100 μl of wash buffer was added to each well to re-suspend the cells, the plate was shaken at 500±50 rpm at room temperature for 2 min.
12) The analysis was conducted with a LuminexTM 100 instrument within 90 min.
1.6 Detection of Serum Cytokines in aGVHD Mice by ELISA Kit
Because lacking the matching mouse TGFβ Luminex kit and ELISA kit, we used the human TGF-β ELISA kit as a substitute reagent in view of the fact that there is high homology between human TGF-β and mouse TGF-β. It is difficult to determine the serum IL-2 concentration. But when a T cell is activated, IL-2 binds to its receptors (with α, β, and γ subunits), and the α subunit falls off of the cell surface and can be detected in the serum, called soluble IL-2 receptor (sIL-2R). Thus, an ELISA kit can be used to re-detect sIL-2R in mice.
The methods for detecting serum sIL-2R in mice and human TGFβ were as follows:
1) The required plate was taken out of the sealed bag after pre-equilibration to room temperature.
2) One well was left blank, and the sample (1:1 dilution) and different concentrations of standard samples (100 μl/well) were added to the other wells. The wells were then sealed with a plate cover and incubated in an incubator at 37° C. for 90 min.
3) The plate was washed 4 times. Except for the blank well, biotin-antibody working solution (100 μl/well) was added to each well. The wells were then sealed with a plate cover and incubated in an incubator at 37° C. for 60 min.
4) The plate was washed 4 times. Except for the blank well, enzyme-conjugating working solution (100 μl/well) was added. The wells were then sealed with a plate cover and incubated in an incubator at 37° C. for 30 min.
5) The plate was washed 4 times. The chromogenic agent (100 μl/well) was added, and then the plate was incubated in an incubator at 37° C. in the dark for 10-15 min.
6) The stop solution (1001/well) was added and mixed well, and OD450 value was measured immediately (within 5 min).
Interpretation of the Results:
CurveExpert 1.3 software was used to graph the standard curve. The concentrations of the standard samples were used as the abscissa, and OD values were used as the ordinates to graph the standard curve. The concentrations of the samples can be determined on the standard curve by using their OD values. If the OD value of a sample was higher than the upper limit of the standard curve, the sample was appropriately diluted for re-measurement. The dilution fold was then multiplied while calculating the concentration of the sample.
1.7 Analysis of Peripheral T-Cell Subpopulations in aGVHD Mice
At week 2 after transplantation, peripheral blood was drawn and treated with heparin sodium. Then, FITC-anti-mouse CD3, PE-anti-mouse CD28, PE-Cy5-anti-mouse CD8, PE-Cy5-anti-mouse CD4, PE-anti-mouse CD4, and PE-anti-mouse CD25 monoclonal antibodies were added. Flow cytometric analysis was conducted to detect changes in the CD4+/CD8+ and CD3+CD8+C28− inhibitory T-cell subpopulations (Ts cells) and the CD4+CD25+ regulatory T-cell subpopulation (Treg cells). The methods were as follows:
1) After 1 μg of corresponding fluorescent-labeled antibodies was added to 100 μl of anticoagulant blood, the sample was incubated at room temperature in the dark for 30 min.
2) Two milliliters of PharmlyseTM erythrocyte lysis buffer was added, followed by incubation at room temperature in the dark for 15 min.
3) The sample was centrifuged at 1500 rpm for 6 min, re-suspended with PBS after the supernatant was discarded, and then centrifuged at 1500 rpm for 5 min again.
4) The supernatant was discarded, and 0.5 ml of 2% paraformaldehyde/PBS was added for detection.
1.8 Statistical Analysis
The data were expressed as x+s, and SPSS 13.0 software was used for statistical analysis. ANOVA and Dunnett's t-test were used for comparison among groups when there was homogeneity of variance. P<0.05 is considered statistically significant. Non-parametric analysis was used when there was heterogeneity of variance.
2. Results
2.1 The Changes of Serum Cytokines in aGVHD Mice after Treatment with B7-1-PE40KDEL DNA Vaccine
On days 7, 14, and 21 after transplantation, Luminex liquid chip and the ELISA method were used to detect the changes of serum cytokines in the mice of all experimental groups. The results are presented in Table 1. The results revealed that among Th1 cytokines, the levels of IFNγ, IL-2, and TNFα were significantly higher in aGVHD mice than in the normal control group (P<0.01), especially during the time period from the beginning stage of the disease's onset to day 7 after transplantation. By day 14 after transplantation, the IFNγ level gradually declined, and by day 21, IFNγ had returned to a near-normal level. In contrast, the levels of IL-2 and TNFα, two of the Th1 cytokines, were maintained at levels far higher than normal. There was no statistical difference in the changes of IL-4 (Th2 cytokine) and TGF-3 (Th3 cytokine) levels between the experimental groups and the normal control group. Surprisingly, on days 7 and 14, the IL-10 (Th2 cytokine) level was significantly higher in aGVHD mice than in the normal control group. Due to the sensitivity of the available detection methods, it was difficult to detect IL-12.
As shown in Table 5, during the 7 days after transplantation, the decline of serum Th1 cytokines such as IFNγ was the most significant (P<0.01) in the mice in B7-1-PE40KDEL DNA vaccine group, B7-2-PE40KDEL DNA vaccine group and B7-1-PE40KDEL+B7-2-PE40KDEL DNA vaccine group. The IFNγ level was still high on day 7 in CsA+MTX group, and the difference was statistically significant when compared with that in the aGVHD group (P<0.01). However, on days 14 and 21, similar to the B7-1-PE40KDEL DNA vaccine group, IFNγ level returned to normal in CsA+MTX group. In addition, IL-2 level was effectively reduced in both B7-1-PE40KDEL DNA vaccine group and CsA+MTX group, and the difference was statistically significant when compared with that in the aGVHD control group (P<0.05). In contrast, the effect on the Th1 cytokine TNF-α was not significant in either B7-1-PE40KDEL DNA vaccine group or CsA+MTX group.
As shown in Table 5, for Th2 cytokines such as IL-4 and IL-10 and Th3 cytokine TGF-β, which are involved in the suppression of inflammation, there was no statistical difference in the effect on Th2 cytokine IL-4 between B7-1-PE40KDEL DNA vaccine group and CsA+MTX group. The levels of Th2 cytokine IL-10 were effectively increased in both B7-1-PE40KDEL DNA vaccine group and CsA+MTX group, and the difference was statistically significant when compared with the level in the aGVHD control group. For the Th3 cytokine TGF-β, only B7-1-PE40KDEL DNA vaccine effectively increased its level, and B7-2-PE40KDEL DNA vaccine group had no significant effect. In contrast, CsA+MTX group slightly decreased the expression level of Th3 cytokine TGF-β.







TABLE 5







The effect of B7-1-PE40KDEL DNA vaccine on Th1, Th2, and Th3 cytokines in the peripheral blood of aGVHD mice









Th1, Th2, and Th3 cytokines (pg/ml)













Group
INF-γ
TNF-α
IL-2
IL-4
IL-10
TGF-β





Normal group + day 7
0
0.52 ± 0.27
11.17 ± 3.47
284.50 ± 22.18
4.70 ± 0.78
1072.55 ± 147.85


+day 14
0
0.52 ± 0.27
11.17 ± 3.47
284.50 ± 22.18
4.70 ± 0.78
1072.55 ± 147.85


+day 21
0
0.52 ± 0.27
11.17 ± 3.47
284.50 ± 22.18
4.70 ± 0.78
1072.55 ± 147.85


NS group + day 7
1465.03 ± 316.77
19.97 ± 3.69 
20.54 ± 1.58
277.94 ± 22.14
8.76 ± 0.53
1239.27 ± 450.30


+day 14
122.37 ± 49.38
11.24 ± 0.73 
23.44 ± 2.52
271.03 ± 22.62
9.99 ± 2.15
1036.73 ± 268.11


+day 21
12.29 ± 8.69
9.64 ± 3.72
23.43 ± 0.89
268.76 ± 29.97
6.88 ± 1.40
1378.67 ± 541.84


Vector group + day 7
1053.63 ± 109.48
15.47 ± 1.25 
29.47 ± 1.58
272.80 ± 17.47
9.29 ± 1.63
1080.59 ± 275.75


+day 14
 73.94 ± 24.60
13.30 ± 1.45 
21.65 ± 1.26
225.88 ± 27.93
10.72 ± 0.88 
1146.16 ± 231.18


+day 21
13.69 ± 6.26
13.15 ± 2.38 
19.65 ± 1.58
238.48 ± 29.56
6.36 ± 1.51
1071.19 ± 88.58 


CsA + MTX + day 7
1874.66 ± 102.67
14.58 ± 0.36 
17.83 ± 2.25
297.65 ± 19.15
11.57 ± 2.29 
 908.40 ± 141.63


+day 14
0
10.43 ± 3.33 
19.39 ± 4.45
232.96 ± 15.66
7.47 ± 1.41
 873.54 ± 136.44


+day 21
0
21.81 ± 6.29 
17.41 ± 3.46
286.30 ± 33.10
10.32 ± 2.62 
 972.62 ± 186.71


B7-1-PE40KDEL + day 7
 533.10 ± 122.13
19.61 ± 1.06 
18.31 ± 1.58
304.99 ± 26.43
12.40 ± 1.88 
1045.65 ± 124.69


+day 14
0
15.58 ± 3.22 
20.09 ± 0.95
268.46 ± 3.60 
12.29 ± 2.51 
1601.20 ± 484.05


+day 21
0
10.43 ± 3.33 
23.44 ± 1.90
255.04 ± 10.23
7.47 ± 0.28
1526.97 ± 402.29


B7-2-PE40KDEL + day 7
 602.20 ± 101.64
20.08 ± 5.27 
14.04 ± 3.12
272.86 ± 26.22
13.03 ± 1.68 
1036.53 ± 76.14 


+day 14
14.63 ± 7.52
17.86 ± 2.13 
17.19 ± 1.90
273.75 ± 20.84
12.08 ± 1.33 
1205.81 ± 122.57


+day 21
0
15.72 ± 1.97 
11.39 ± 2.52
255.88 ± 18.82
12.50 ± 1.33 
1363.53 ± 366.11


B7-1 + B7-2-PE40KDEL + day 7
 419.59 ± 137.29
16.73 ± 0.18 
15.85 ± 8.20
261.58 ± 18.46
11.97 ± 0.63 
1322.33 ± 245.55


+day 14
0
12.14 ± 1.28 
 3.18 ± 5.68
257.22 ± 10.24
10.75 ± 3.21 
1256.07 ± 368.46


+day 21
0
17.35 ± 12.13
21.65 ± 1.26
247.09 ± 15.32
9.49 ± 0.29
 847.32 ± 155.79









2.2 The Changes of Peripheral T-Cell Subpopulations in aGVHD Mice after Treatment with the B7-1-PE40KDEL DNA Vaccine
During week 2 after transplantation, flow cytometric analysis was conducted to detect three T-cell subpopulations (i.e., CD4+/CD8+, CD4+CD25+Treg and CD8+C28−Ts subpopulations). The results and representative photos are presented in FIGS. 26-31 and Table 6.
The results indicated that in the normal group, CD4/CD8 ratio was approximately 3.2, which was seriously inverted in aGVHD mice after radiation and transplantation. In aGVHD mice after radiation and transplantation compared with the normal group, CD3+CD8+T-cell proportions were significantly increased, whereas CD3+CD4+ proportions were significantly decreased. B7-1-PE40KDEL DNA vaccine and B7-2-PE40KDEL DNA vaccines, B7-1-PE40KDEL+B7-2-PE40KDEL DNA vaccine, and CsA+MTX treatment did not significantly reverse CD4/CD8 ratio (FIGS. 26-27 and Table 6).
To determine whether specific T cell subpopulations participate in immune regulation in the B7-1-PE40KDEL DNA vaccine group and CsA+MTX group, we compared the changes of CD4+CD25+Treg proportions among CD4+ lymphocytes in each group and the control group (FIGS. 28-29 and Table 5). The results revealed that CD4+CD25+Treg proportions were significantly higher in the B7-1-PE40KDEL DNA vaccine group and B7-1-PE40KDEL+B7-1-PE40KDEL DNA vaccines group than in the empty vector group and the NS control group (P<0.01 & P<0.05). The CD4+CD25+Treg proportions were also higher in B7-2-PE40KDEL DNA vaccine group than in the empty vector group and the NS control group (P<0.05), but the effect of B7-2-PE40KDEL DNA vaccine was weaker than that of B7-1-PE40KDEL DNA vaccine. In contrast, there was no significant difference in CD4+CD25+Treg proportions in CsA+MTX group.
In addition, we also compared the changes in the regulation of the immune network by another regulatory T-cell (i.e., the changes in CD8+CD28−Ts proportions among all groups). The results are shown in FIGS. 30-31 and Table 6. It can be seen that CD8+CD28−Ts proportions were higher in B7-1-PE40KDEL DNA vaccine, B7-2-PE40KDEL DNA vaccine, B7-1-PE40KDEL+B7-2-PE40KDEL DNA vaccine and CsA+MTX groups than in the empty vector group and the NS control group, and the differences were significant (P<0.01).







TABLE 6







The effect of B7-1-PE40KDEL DNA vaccine on CD4+CD25+ and


CD8+CD28−T sub-groups and CD4/CD8 ratios in the peripheral blood of


aGVHD mice










Group
% CD4+CD25+T
% CD8+CD28−T
CD4/CD8 Ratio














Normal group
+ day 7
1.53 ± 0.60
14.46 ± 1.50 
3.25 ± 0.84



+ day 14
1.53 ± 0.60
14.46 ± 1.50 
3.25 ± 0.84



+ day 21
1.53 ± 0.60
14.46 ± 1.50 
3.25 ± 0.84


NS group
+ day 7
2.04 ± 1.05
0.89 ± 0.94
0.31 ± 0.02



+ day 14
1.25 ± 0.34
9.18 ± 6.34
0.23 ± 0.01



+ day 21
1.32 ± 0.40
7.25 ± 2.18
0.42 ± 0.09


Vector group
+ day 7
1.38 ± 0.59
0.69 ± 0.12
0.31 ± 0.01



+ day 14
1.31 ± 0.44
14.64 ± 1.25 
0.21 ± 0.04



+ day 21
1.32 ± 0.43
5.66 ± 2.53
0.42 ± 0.07


CsA + MTX
+ day 7
1.55 ± 1.06
1.71 ± 0.66
0.27 ± 0.04



+ day 14
1.27 ± 0.27
14.39 ± 1.71 
0.24 ± 0.03



+ day 21
1.38 ± 1.05
6.33 ± 2.93
0.45 ± 0.04


B7-1-PE40KDEL
+ day 7
1.73 ± 0.72
2.01 ± 0.24
0.34 ± 0.04



+ day 14
2.22 ± 0.50
18.76 ± 6.21 
0.19 ± 0.02



+ day 21
4.88 ± 4.18
5.71 ± 1.01
0.44 ± 0.11


B7-2-PE40KDEL
+ day 7
1.11 ± 0.51
2.55 ± 0.64
0.27 ± 0.03



+ day 14
1.93 ± 1.21
22.67 ± 6.74 
0.20 ± 0.03



+ day 21
1.39 ± 0.36
9.54 ± 2.94
0.39 ± 0.02


B7-1 + B7-2-PE40KDEL
+ day 7
2.59 ± 2.04
2.51 ± 0.34
0.27 ± 0.01



+ day 14
1.94 ± 0.14
20.20 ± 5.44 
0.20 ± 0.01



+ day 21
1.96 ± 0.11
6.41 ± 3.00
0.29 ± 0.05









The invention includes the following non-limited embodiments:
1. A gene which encodes an exotoxin fusion protein B7-1-PE40KDEL, wherein the nucleotide sequence of said gene is
(1) A sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2,
(2) A sequence which encodes the same protein encoded by the sequences shown in SEQ ID NO:1 or SEQ ID NO:2, and the nucleotide sequences differs from the sequences shown in SEQ ID NO:1 or SEQ ID NO:2 due to the codon degeneracy,
(3) A sequence which hybridizes with the sequences shown in SEQ ID NO:1 or SEQ ID NO:2 under strict hybridizing conditions, and the protein encoded by said sequence having the same or similar function as the protein encoded by the sequence shown in SEQ ID NO:1 or SEQ ID NO:2, or
(4) A sequence having more than 75% identity with the sequence shown in SEQ ID NO:1 or SEQ ID NO:2, preferably, more than 85%.
2. An exotoxin fusion protein which is encoded by the gene according to embodiment 1.
3. A recombinant expression vector which is operationally linked to the gene according to embodiment 1, and said expression vector is selected from eukaryotic expression vectors or prokaryotic expression vectors, preferably, said gene is operationally linked to anyone of the expression vectors selected from pcDNA3.1/Zeo(+), pWLNEO, pSV2CAT, pOG44, pXT1, pSG, pSVK3, pBPV, pMSG, pSVL, and adenovirus.
4. The recombinant expression vector according to embodiment 3, which consists of the gene of embodiment 1 and the pcDNA3.1/Zeo(+) vector.
5. A composition which contains the gene according to embodiment 1, the exotoxin fusion protein according to embodiment 2, or the recombinant expression vector according to embodiment 3 or 4.
6. A vaccine, which contains the gene according to embodiment 1, the exotoxin fusion protein according to embodiment 2, or the recombinant expression vector according to embodiment 3 or 4, and the pharmaceutical acceptable adjuvant.
7. A DNA vaccine which comprises the recombinant expression vector according to embodiment 3 or 4, and the pharmaceutically acceptable immune adjuvants.
8. A formulation of the composition according to embodiment 5, the vaccine according to embodiment 6, or the DNA vaccine according to embodiment 7, which be prepared in the form are suitable for the administration via intravenous injection, intra-arterial injection, intramuscular injection, subcutaneous injection, organ injection, intra-pleural injections, and intraperitoneal injection.
9. The composition according to embodiment 5, the vaccine according to embodiment 6, or the DNA vaccine according to embodiment 7, which be formulated in the form of an aqueous solution or a reconstituted freeze-dried powder, can be use in the way of injection or administration via the mucosa.
10. A method for treating or preventing allogeneic tissue and/or organ transplant rejections, comprise to administrate the composition according to embodiment 5, the vaccine according to embodiment 6, or the DNA vaccine according to embodiment 7 in an effective amount to the subject in need thereof.
11. A use of the composition according to embodiment 5, the vaccine according to embodiment 6, or the DNA vaccine according to embodiment 7 in the preparation of a medicine for treating or preventing allogeneic tissue and/or organ transplant rejections.
The embodiments of the invention have been described in detail and will be understood by a person skilled in the art. The details can be modified and substituted according to the published literature. These changes are within the scope of protection of this invention. The full scope of the invention is provided by the appended claims and any equivalents thereof.

## Claims

1. A non-naturally occurring DNA construct encoding exotoxin fusion protein B7-1-PE40KDEL
that comprises a polynucleotide sequence having at least 95% identity to the entire length of SEQ ID NO: 1 or at least 95% identity to the entire length SEQ ID NO: 2; or a polynucleotide sequence that encodes the same protein as the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and
that encodes an exotoxin fusion protein that when administered to a mammal immunosuppresses T cells.

2. An exotoxin fusion protein encoded by the DNA construct of claim 1.

3. A recombinant expression vector which is operably-linked to the DNA construct of claim 1.

4. The recombinant expression vector of claim 3, which when transfected into a eukaryotic cell is transcribed and translated into a polypeptide that is secreted extracellularly.

5. A composition comprising the DNA construct of claim 1.

6. A composition comprising the exotoxin fusion protein of claim 2 and a pharmaceutical acceptable adjuvant.

7. A composition comprising the recombinant expression vector of claim 3, and a pharmaceutically acceptable immune adjuvant.

8. A formulation of the composition of claim 5, which is suitable for administration via intravenous injection, intra-arterial injection, intramuscular injection, subcutaneous injection, organ injection, intra-pleural injection, or intraperitoneal injection.

9. The composition of claim 5, in a form of an aqueous solution or a reconstituted freeze-dried powder, suitable for injection or administration via a mucosa.

10. A method for treating or inhibiting rejection of an allogeneic tissue or an organ transplant, the method comprising administering the composition of claim 5 in an effective amount to a subject in need thereof.

11. The DNA construct of claim 1 comprising the sequence of SEQ ID NO: 1.

12. The DNA construct of claim 1, comprising the sequence of SEQ ID NO: 2.

13. The DNA construct of claim 1, comprising a nucleotide sequence that encodes the same protein as that encoded by the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, wherein the nucleotide sequence differs from SEQ ID NO: 1 or SEQ ID NO: 2 due to codon degeneracy.

14. An expression vector comprising the DNA construct of claim 1 and plasmid pcDNA3.1/Zeo(+) that when transfected into a eukaryotic cell is transcribed and translated into a polypeptide that is secreted extracellularly.

15. A composition comprising the expression vector of claim 14 and a pharmaceutically acceptable adjuvant.

16. Expression vector pcDNA3.1/Zeo(+)-B7-1-PE40KDEL.

17. A composition comprising the expression vector of claim 16 and a pharmaceutically acceptable adjuvant.

18. An isolated gene encoding exotoxin fusion protein B7-1-PE40KDEL that comprises:
(1) the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or
(2) a nucleotide sequence encoding the same exotoxin fusion protein encoded by the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2, wherein the nucleotide sequence differs from SEQ ID NO:1 or SEQ ID NO:2 due to codon degeneracy.

19. A method for treating graft-vs-host disease, host-vs-graft disease or another condition mediated by activation of T cells comprising administering to a subject in need thereof the non-naturally-occurring DNA construct of claim 1.