Immunomodulation of Transgene Responses Following Naked DNA Transfer of Factor VIII into Hemophilia A Mice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1279-1279
Author(s):  
Peiqing Ye ◽  
David J. Rawlings ◽  
Arthur R. Thompson ◽  
Hans D. Ochs ◽  
Carol H. Miao
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Immune Responses ◽  
Factor Viii ◽  
Hemophilia A ◽  
Dna Transfer ◽  
Secondary Response ◽  
Inhibitory Antibody ◽  
Naked Dna ◽  
Human Factor Viii

Abstract Naked DNA transfer of liver-specific, high-expressing plasmid pBS-HCRHPI-FVIIIA in Rag2(−/ −) SCID mice produced persistent high-level gene expression of human factor VIII (hFVIII) (Miao, Hum. Gene Ther. 2003). However, in immunocompetent hemophilia A mice, a robust humoral immune response against FVIII that followed gene transfer led to complete inhibition of circulating FVIII activity (Ye, Mol. Ther. 2004). Transient immunomodulation strategies were explored to prevent the formation of inhibitory antibody formation. Eight groups of mice (n=8) were treated by naked DNA transfer of plasmid pBS-HCRHPI-FVIIIA. Each group were subjected to treatment with single or combined immunosuppressive regimen: CyclosporineA (CSA) daily for 14 days; Rapamycin daily for 14 days; Mycophenylate mofetil (MMF) daily for 14 days; combination of CSA and MMF; combination of Rapamycin and MMF; a monoclonal antibody (MR1) against murine CD40 ligand on days -1, 1, 2, 7, & 14; recombinant murine Ctla4Ig on days 1 & 2; and combination of MR1 and Ctla4Ig. Combination regimens were given using the same combined schedule and dosages. All animals treated with immunosuppression had delayed or no immune responses against hFVIII except the group treated with CSA only. The most effective treatment was observed in animals treated with the combination of Ctla4Ig and MR1. Seven of 8 animals failed to develop detectable inhibitors. One animal developed transient low-titer antibodies. This group of animals produced persistent, therapeutic levels of hFVIII gene expression for over 6 months. Tolerized animals were subsequently challenged by the T dependent antigen, bacteriophage Φx174, and exhibited a normal primary and secondary response including amplification and isotype switch. These results strongly suggest that transient immunomodulation strategies to disrupt B- and T- cell interactions at the time of plasmid injection is effective to promote long-term immune tolerance that is specific for FVIII without altering subsequent immune responses to other T cell dependent antigens.

Immune Tolerance to Human Factor VIII Induced by Liver-Specific Expression of a Human Factor VIII Transgene in Hemophilic Mice.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3387-3387 ◽  
Author(s):  
Pauline van Helden ◽  
Maria Sasgary ◽  
Sabine Unterthurner ◽  
Maria Schuster ◽  
Gerhard Antoine ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Immune Responses ◽  
Factor Viii ◽  
Immune Tolerance ◽  
Preliminary Data ◽  
Human Factor ◽  
Hemophilia A ◽  
Specific Expression ◽  
Human Factor Viii

Abstract Therapy of hemophilia A has greatly benefited from the development of safe recombinant and plasmatic factor VIII (FVIII) concentrates. Current efforts to improve products focus on the extension of half-life by chemical and/or molecular modifications of FVIII. However, any modification of the FVIII protein poses the risk of creating neo-antigens that might cause FVIII inhibitors to be induced in patients. Therefore it is important to monitor the potential creation of neo-antigens during preclinical and clinical phases of drug development. Currently available animal models for hemophilia A develop high titers of anti-FVIII antibodies when treated with human FVIII. Using these models, it is difficult to differentiate between immune responses against native human FVIII and immune responses against human FVIII that carries neo-antigens. Considering these limitations, our aim is to develop a new model for hemophilia A that does not respond with antibodies to native human FVIII but develops antibodies against human FVIII that carries neo-antigens. We created a series of hemophilic mouse lines that carry a transgene for human FVIII that was placed under the control of an albumin promoter to direct liver-specific expression. Transgenic founder mice were generated by direct microinjection of the vector into the male pronucleus of fertilized oocytes obtained from mated female C57BL/6J mice after superovulation. Transgenic mice were crossed with hemophilic mice and bred to homozygousity for the expression of the human FVIII transgene. We analyzed the expression of human FVIII by real time PCR in lung, kidney, liver, heart, muscle, spleen, lymph nodes and reproductive organs. Gene expression analysis of bone marrow and thymus are currently ongoing. We selected three sublines (E, G and I) that show different levels of liver-specific expression of human FVIII for further analysis. We did not detect any FVIII antigen in the circulation in any of these three sublines when we used two different ELISA systems with detection limits around 1 ng/ml. We treated mice of sublines E, G and I intravenously with eight weekly doses of 200 ng of human FVIII (Advate) and analyzed the potential development of antibodies against native human FVIII. Our results indicate that transgenic mice of sublines E and I are immunologically tolerant to native human FVIII. They do not develop anti-FVIII antibodies (about 90% of all mice tested) or develop low titers (below 1:80 in 10% of mice tested) only. In contrast, mice of subline G develop high titers of anti-FVIII antibodies indicating that they are not immunologically tolerant to human FVIII. Preliminary data suggest that the degree of immunological tolerance against human FVIII correlates to a certain extent with the expression levels of the human FVIII transgen in liver and/or thymus. We are in the process of verifying these preliminary data. Furthermore, we have started to analyze FVIII-specific T-cell responses to define potential differences in the repertoire of FVIIIspecific T cells between the three sublines. We conclude that transgenic expression of human FVIII under the control of an albumin promoter is able to induce immune tolerance to human native FVIII in hemophilic mice. However, a certain threshold level of gene expression might be required for the induction of immune tolerance.

scholarly journals Immunomodulation of transgene responses following naked DNA transfer of human factor VIII into hemophilia A mice

Blood ◽  
2006 ◽  
Vol 108 (1) ◽  
pp. 19-27 ◽  
Author(s):  
Carol H. Miao ◽  
Peiqing Ye ◽  
Arthur R. Thompson ◽  
David J. Rawlings ◽  
Hans D. Ochs
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Hemophilia A ◽  
Cd40 Ligand ◽  
Cell Interaction ◽  
Dna Transfer ◽  
Humoral Immune ◽  
Naked Dna ◽  
Human Factor Viii ◽  
High Level

A robust humoral immune response against human factor VIII (hFVIII) following naked DNA transfer into immunocompetent hemophilia A mice completely inhibits circulating FVIII activity despite initial high-level hFVIII gene expression. To prevent this undesirable response, we compared transient immunomodulation strategies. Eight groups of mice (n = 4-9 per group) were treated with naked DNA transfer of pBS-HCRHPI-hFVIIIA simultaneously with immunosuppressive reagents that included cyclosporine A (CSA), rapamycin (RAP), mycophenylate mofetil (MMF), a combination of CSA and MMF, a combination of RAP and MMF, a monoclonal antibody against murine CD40 ligand (MR1), recombinant murine Ctla4Ig, and a combination of MR1 and Ctla4Ig. All animals except those receiving only CSA exhibited delayed or absent immune responses against hFVIII. The most effective immunosuppressive regimen, the combination of Ctla4Ig and MR1, prevented inhibitor formation in 8 of 9 animals; the ninth had transient low-titer antibodies. All 9 mice of this group produced persistent, therapeutic levels of hFVIII for more than 6 months. When challenged with the T-dependent antigen bacteriophage Φx174, tolerized mice exhibited normal primary and secondary antibody responses, suggesting that transient immunomodulation to disrupt B/T-cell interaction at the time of plasmid injection effectively promoted long-term immune tolerance specific for hFVIII. (Blood. 2006;108:19-27)

Reduction of the inhibitory antibody response to human factor VIII in hemophilia A mice by mutagenesis of the A2 domain B-cell epitope

Blood ◽  
2004 ◽  
Vol 104 (3) ◽  
pp. 704-710 ◽  
Author(s):  
Ernest T. Parker ◽  
John F. Healey ◽  
Rachel T. Barrow ◽  
Heather N. Craddock ◽  
Pete Lollar
Keyword(s):  
Antibody Response ◽  
Factor Viii ◽  
B Cell ◽  
Human Factor ◽  
Hemophilia A ◽  
Cell Epitope ◽  
Inhibitory Antibody ◽  
B Cell Epitope ◽  
Human Factor Viii ◽  
Inhibitory Antibodies

AbstractApproximately 25% of patients with hemophilia A develop inhibitory antibodies after treatment with factor VIII. Most of the inhibitory activity is directed against epitopes in the A2 and C2 domains. Anti-A2 inhibitory antibodies recognize a 25-residue segment bounded by R484-I508. Several antigenic residues in this segment have been identified, including R484, R489, and P492. The immunogenicity of purified recombinant B domain–deleted (BDD) human factor VIII molecules containing mutations at R484A/R489A or R484A/R489A/P492A was studied in hemophilia A mice. Inhibitory antibody titers in mice receiving the R484A/R489A/P492A mutant, but not the R484A/R489A mutant, were significantly lower than in mice receiving control human BDD factor VIII. The specific coagulant activity and the in vivo clearance and hemostatic efficacy in hemophilia A mice of the R484A/R489A/P492A mutant were indistinguishable from human BDD factor VIII. Thus, the inhibitory antibody response to human factor VIII can be reduced by mutagenesis of a B-cell epitope without apparent loss of function, suggesting that this approach may be useful for developing a safer form of factor VIII in patients with hemophilia A.

Humanized E17 Hemophilic Mice Are a Major Breakthrough in the Design of New Preclinical Models for Developing Factor VIII Products with Reduced Immunogenicity.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 782-782 ◽  
Author(s):  
Birgit M. Reipert ◽  
Christina Hausl ◽  
Maria Sasgary ◽  
Maria Schuster ◽  
Rafi U. Ahmad ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Factor Viii ◽  
Mhc Class Ii ◽  
Cd4 T Cells ◽  
Plasma Cells ◽  
Hemophilia A ◽  
Class Ii ◽  
Mhc Class Ii Molecules

Abstract MHC class II molecules are crucial for regulating adaptive immune responses against self and foreign protein antigens. They determine the antigenic peptides that are presented to CD4+ T cells and are essential for shaping the CD4+ T-cell repertoire in the thymus. Thus, the structure of MHC class II molecules is a major determinant for protein antigen immunogenicity. Structural differences between murine and human MHC class II complexes fundamentally limit the use of conventional murine hemophilia A models for dissecting immune responses to human factor VIII and developing new factor VIII products with reduced immunogenicity. To overcome this limitation, we humanized the murine E17 model of hemophilia A by introducing the human MHC class II haplotype HLA-DRB1*1501 on the background of a complete knockout of all murine MHC class II genes. Any anti-FVIII antibody response in this new humanized hemophilia A model is driven by CD4+ T cells that recognize FVIII-derived peptides that are presented by human HLA-DRB1*1501. The MHC class II haplotype HLA-DRB1*1501 is particularly relevant for the situation in hemophilia A patients because it is found in about 25% of Caucasians and 32% of Africans and has been shown to be associated with an increased risk that patients with severe hemophilia A have for developing FVIII inhibitors. We validated the relevance of this new model by asking the question whether HLA-DRB1*1501 hemophilic E17 mice develop FVIII inhibitors that are similar to those observed in patients with hemophilia A. Furthermore, we wanted to show that anti-FVIII antibody responses in these mice depend on the expression of the human DRB1*1501 molecule. Mice were treated with 8 intravenous doses of human FVIII and tested for anti-FVIII antibodies, anti-FVIII antibody-producing plasma cells and FVIII-specific T cells. About 90% of all humanized hemophilic E17 mice tested developed anti-FVIII antibodies that were similar to FVIII inhibitors found in patients. These antibodies were not restricted isotypically and contained mainly IgG1, IgG2a and IgG2b antibodies. Detection of antibodies in the circulation correlated with the presence of anti-FVIII antibody-producing plasma cells in the spleen. Development of anti-FVIII antibodies depended on the activation of FVIII-specific T cells and strictly depended on the expression of the HLA-DRB1*1501 molecule. Mice that did not express any MHC class II molecules did not develop anti-FVIII antibodies. We conclude that this new humanized E17 model for hemophilia A is a major advance towards developing suitable animal models needed to design future immunomodulatory strategies for patients with FVIII inhibitors and develop new FVIII products with reduced immunogenicity. Furthermore, it provides a tool for identifying T-cell epitopes of human FVIII restricted by MHC class II molecules that can be used for monitoring FVIII-specific T cells in patients who receive replacement therapy with FVIII products.

scholarly journals Naked DNA Transfer of Factor VIII Induced Transgene-Specific, Species-Independent Immune Response in Hemophilia A Mice

2004 ◽  
Vol 10 (1) ◽  
pp. 117-126 ◽  
Author(s):  
Peiqing Ye ◽  
Arthur R. Thompson ◽  
Rita Sarkar ◽  
Zhenping Shen ◽  
David P. Lillicrap ◽  
...  
Keyword(s):  
Immune Response ◽  
Factor Viii ◽  
Hemophilia A ◽  
Dna Transfer ◽  
Naked Dna ◽  
Specific Species

Immunodominant T-cell epitopes in the factor VIII C2 domain are located within an inhibitory antibody binding site

2004 ◽  
Vol 92 (09) ◽  
pp. 522-528 ◽  
Author(s):  
David Okita ◽  
Brenda Diethelm-Okita ◽  
Bianca Conti-Fine ◽  
Kathleen Pratt ◽  
Jiahua Qian ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Factor Viii ◽  
Human Monoclonal Antibody ◽  
Cell Epitope ◽  
Major Complication ◽  
C2 Domain ◽  
T Cell Epitopes ◽  
Inhibitory Antibody ◽  
Human Factor Viii

SummaryFormation of inhibitor antibodies to factor VIII (FVIII) is a major complication of FVIII replacement therapy for hemophilia A patients, and it occurs through a T-cell dependent process. The C2 domain of FVIII contains epitopes that are recognized by antibody inhibitors. We have examined regions of the C2 domain that form epitopes for T cells in mice congenitally deficient in FVIII. We obtained CD4+ T cells from mice immunized by intravenous infusion of therapeutic doses of recombinant human FVIII (rFVIII), or by subcutaneous injections of rFVIII or recombinant human C2 domain in adjuvant. In all cases, the T cells recognized most strongly and consistently two overlapping peptides that spanned residues 2191 to 2220 of the C2 domain. Analysis of the crystal structure of human factor VIII C2 bound to a human monoclonal antibody, BO2C11, showed these residues also constitute part of a human alloimmune B-cell epitope (Spiegel et al., Blood 2001; 98: 13-19).This region includes one of the “hydrophobic spike” protrusions, consisting of M2199 and F2200, as well as the basic residues R2215 and R2220. These residues contribute to membrane binding and to association with von Willebrand factor (vWF).These findings suggest that a major T-cell epitope in the C2 domain recognized by hemophilic mice is located within the same region that binds to inhibitors, vWF, and activated membranes.

Prevention and treatment of factor VIII inhibitors in murine hemophilia A

Blood ◽  
2000 ◽  
Vol 95 (4) ◽  
pp. 1324-1329 ◽  
Author(s):  
Jiahua Qian ◽  
Mary Collins ◽  
Arlene H. Sharpe ◽  
Leon W. Hoyer
Keyword(s):  
T Cell ◽  
Factor Viii ◽  
Antibody Formation ◽  
Hemophilia A ◽  
Major Complication ◽  
Primary Response ◽  
Inhibitory Antibody ◽  
Intravenous Injections ◽  
Antibody Titers ◽  
Viii Inhibitor

Inhibitory antibody formation is a major complication of factor VIII replacement therapy in patients with hemophilia A. To better understand the pathogenesis of this immunologic reaction, we evaluated the role of T-cell costimulatory signals for antifactor VIII antibody formation in a murine model of hemophilia A. Repeated intravenous injections of factor VIII in these factor VIII–deficient mice induced an antifactor VIII inhibitor antibody response. This response was shown to be T-cell dependent by its absence in hemophilic mice also deficient for the T-cell costimulatory ligand B7-2. In separate experiments, injection of murine CTLA4-Ig completely blocked the primary response to factor VIII in hemophilic mice with intact B7 function. This reagent also prevented or diminished further increases in antifactor VIII when given to hemophilic mice with low antifactor VIII antibody titers. These studies suggest that strategies targeting the B7-CD28 pathway are potential therapies to prevent and treat inhibitory antifactor VIII antibodies. Moreover, because the development of antibodies to replaced proteins may limit the success of many human gene therapy approaches, our results may be broadly applicable.

Restricted BV gene usage by factor VIII-reactive CD4+ T cells in inhibitor-positive patients with severe hemophilia A

2003 ◽  
Vol 90 (11) ◽  
pp. 813-822 ◽  
Author(s):  
Jagadeesh Bayry ◽  
Anastas Pashov ◽  
Srini Kaveri ◽  
Roseline d’Oiron ◽  
Natalie Stieltjes ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Factor Viii ◽  
Cd4 T Cells ◽  
Hemophilia A ◽  
Cell Receptor ◽  
Severe Hemophilia ◽  
Cell Repertoire ◽  
Human Factor Viii ◽  
Gene Usage

SummaryIn the present study, we have analyzed the T cell receptor (TCR) repertoires of CD4+ T cells isolated from peripheral blood of 10 inhibitor-positive patients with severe hemophilia A. The distribution of complementarity determining region (CDR3) lengths of the beta chain of the TCRs was analyzed by spectratyping prior to and following in vitrostimulation of the cells with human factor VIII (FVIII). The repertoires of CD4+ T cells of patients were perturbed when compared to those of healthy blood donors. The perturbations of T cell repertoires were heterogeneous among patients with respect to the number and the nature of V-beta (BV) families that exhibited expansion following incubation with FVIII. Some patients showed alterations in one or two BV families, others exhibited more perturbed repertoires affecting 5 to 8 of the 14 BV families tested. Alterations of BV2, BV5 and/or BV9 were consistently found after incubation of CD4+ T cells in the presence of FVIII in 80% of the patients. These findings indicate that the presence of FVIII inhibitors in patients with severe hemophilia A is associated with measurable perturbations of the CD4+ T cell repertoire that results from oligoclonal expansion of FVIII-specific cells and may be relevant for the design of strategies aimed at modulating the anti-FVIII immune responses by T cell-targeted therapy

scholarly journals Lineages of human T-cell clones, including T helper 17/T helper 1 cells, isolated at different stages of anti–factor VIII immune responses

Blood ◽  
2009 ◽  
Vol 114 (7) ◽  
pp. 1423-1428 ◽  
Author(s):  
Ruth A. Ettinger ◽  
Eddie A. James ◽  
William W. Kwok ◽  
Arthur R. Thompson ◽  
Kathleen P. Pratt
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Factor Viii ◽  
Hemophilia A ◽  
T Helper ◽  
T Cell Clones ◽  
Human T Cell ◽  
Cell Clones ◽  
Polarized Cells

AbstractThe development of neutralizing antibodies (inhibitors) after factor VIII (FVIII) infusions is a serious complication that affects approximately one-quarter of hemophilia A patients who have access to replacement therapy. To investigate the differentiation of naive T cells into FVIII-specific helper T cells that promote B-cell activation and antibody secretion, HLA-DRA-DRB1*0101-restricted T-cell clones that respond to a specific epitope in FVIII were isolated from a mild hemophilia A subject (the proband) 19 weeks and 21 months after his development of a high-titer inhibitor. Clones responding to the same epitope were also isolated from his multiply infused brother, who has not developed a clinically significant inhibitor. The 19-week proband clones were T helper (TH)17/TH1- or TH1/TH2-polarized, whereas all 8 clones isolated 21 months postinhibitor development were TH2-polarized cells. In contrast, all 6 clones from the brother who did not develop an inhibitor were TH1-polarized, indicating that tolerance to FVIII can be maintained even with circulating TH1-polarized cells that respond vigorously to in vitro FVIII stimulation. This is the first evidence that TH17/TH1-polarized cells play a role in hemophilic immune responses to FVIII. Furthermore, this is the first report of successful isolation and expansion of antigen-specific human TH17/TH1 clones using standard culture conditions.

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