ovalbumin and Hemophilia-A

Despite >80 years of clinical experience with coagulation factor VIII (FVIII) inhibitors, surprisingly little is known about the in vivo mechanism of this most serious complication of replacement therapy for hemophilia A. These neutralizing antidrug alloantibodies arise in ∼30% of patients. Inhibitor formation is T-cell dependent, but events leading up to helper T-cell activation have been elusive because of, in part, the complex anatomy and cellular makeup of the spleen. Here, we show that FVIII antigen presentation to CD4+ T cells critically depends on a select set of several anatomically distinct antigen-presenting cells, whereby marginal zone B cells and marginal zone and marginal metallophilic macrophages but not red pulp macrophages (RPMFs) participate in shuttling FVIII to the white pulp in which conventional dendritic cells (DCs) prime helper T cells, which then differentiate into follicular helper T (Tfh) cells. Toll-like receptor 9 stimulation accelerated Tfh cell responses and germinal center and inhibitor formation, whereas systemic administration of FVIII alone in hemophilia A mice increased frequencies of monocyte-derived and plasmacytoid DCs. Moreover, FVIII enhanced T-cell proliferation to another protein antigen (ovalbumin), and inflammatory signaling-deficient mice were less likely to develop inhibitors, indicating that FVIII may have intrinsic immunostimulatory properties. Ovalbumin, which, unlike FVIII, is absorbed into the RPMF compartment, fails to elicit T-cell proliferative and antibody responses when administered at the same dose as FVIII. Altogether, we propose that an antigen trafficking pattern that results in efficient in vivo delivery to DCs and inflammatory signaling, shape the immunogenicity of FVIII.

Topics: Animals; CD4-Positive T-Lymphocytes; Dendritic Cells; Factor VIII; Hemophilia A; Hemostatics; Mice; Ovalbumin

A major complication with enzyme replacement therapy of Factor VIII (FVIII) in Hemophilia A (HA) is the development of anti-drug antibodies. Recently, we have shown that FVIII administration in the presence of heterogeneous phosphatidylserine (PS) nanoparticles derived from a natural source induces tolerance to FVIII, suggesting that PS converts an immunogen to a tolerogen. However, the specific structural features responsible for the immune-regulatory properties of PS is unclear. Identifying a specific PS species that is responsible is critical in order to further develop and optimize this nanoparticle. Further, clinical development of this lipid-based strategy requires optimization of the lipid particle that is homogeneous and synthetic. Here, we investigate the ability of mono-acylated Lyso-PS to induce hypo-responsiveness towards FVIII in HA mice. Administration of both PS and Lyso-PS FVIII significantly reduced anti-FVIII antibody responses despite rechallenge with FVIII. Additionally, the Lyso-PS-mediated effect was shown to be antigen-specific as mice responded normally against a rechallenge with an unrelated antigen, ovalbumin. Furthermore, the hypo-responsiveness observed with Lyso-PS may involve interactions with a specific PS receptor, TIM-4, along with increasing regulatory T-cells. These data indicate that using Lyso-PS allows for a more homogenous formulation in order to induce tolerance towards therapeutic proteins.

Topics: Animals; Antibodies; Disease Models, Animal; Factor VIII; Hemophilia A; Immune Tolerance; Injections, Subcutaneous; Membrane Proteins; Mice, Transgenic; Nanoparticles; Ovalbumin; Phosphatidylserines; T-Lymphocytes, Regulatory

Current treatments to control pathological or unwanted immune responses often use broadly immunosuppressive drugs. New approaches to induce antigen-specific immunological tolerance that control both cellular and humoral immune responses are desirable. Here we describe the use of synthetic, biodegradable nanoparticles carrying either protein or peptide antigens and a tolerogenic immunomodulator, rapamycin, to induce durable and antigen-specific immune tolerance, even in the presence of potent Toll-like receptor agonists. Treatment with tolerogenic nanoparticles results in the inhibition of CD4+ and CD8+ T-cell activation, an increase in regulatory cells, durable B-cell tolerance resistant to multiple immunogenic challenges, and the inhibition of antigen-specific hypersensitivity reactions, relapsing experimental autoimmune encephalomyelitis, and antibody responses against coagulation factor VIII in hemophilia A mice, even in animals previously sensitized to antigen. Only encapsulated rapamycin, not the free form, could induce immunological tolerance. Tolerogenic nanoparticle therapy represents a potential novel approach for the treatment of allergies, autoimmune diseases, and prevention of antidrug antibodies against biologic therapies.

Topics: Animals; Antigens; CD4-Positive T-Lymphocytes; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Factor VIII; Female; Hemocyanins; Hemophilia A; Humans; Hypersensitivity, Delayed; Immune Tolerance; Immunity, Humoral; Immunosuppression Therapy; Immunosuppressive Agents; Lactic Acid; Mice; Mice, Inbred BALB C; Nanocapsules; Nanoparticles; Oligodeoxyribonucleotides; Ovalbumin; Peptide Fragments; Peptides; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Proteins; Recombinant Proteins; Sirolimus

Administration of human factor VIII (FVIII) to FVIII knockout hemophilia mice is a useful small animal model to study the physiologic response in patients iatrogenically immunized to this therapeutic protein. These mice manifest a robust, T cell-dependent, antibody response to exogenous FVIII treatment, even when encountered through traditionally tolerogenic routes. Thus, FVIII given via these routes elicits both T- and B-cell responses, whereas a control, foreign protein, such as ovalbumin (OVA), is poorly immunogenic. When FVIII is heat inactivated, it loses function and much of its immunogenicity. This suggests that FVIII's immunogenicity is principally tied to its function and not its structure. If mice are treated with the anticoagulant warfarin, which depletes other coagulation factors including thrombin, there is a reduced immune response to FVIII. Furthermore, when mice are treated with the direct thrombin inhibitor, hirudin, the T-cell responses and the serum anti-FVIII antibody concentrations are again significantly reduced. Notably, when FVIII is mixed with OVA, it acts to increase the immune response to OVA. Finally, administration of thrombin with OVA is sufficient to induce immune responses to OVA. Overall, these data support the hypothesis that formation of thrombin through the procoagulant activity of FVIII is necessary to induce costimulation for the immune response to FVIII treatment.

Topics: Adjuvants, Immunologic; Animals; Anticoagulants; Factor VIII; Hemophilia A; Mice; Mice, Knockout; Ovalbumin; Thrombin

Hemophilia A patients are typically treated by factor VIII (FVIII) protein replacement, an expensive therapy that induces FVIII-specific inhibitors in approximately 30% of patients with severe hemophilia. FVIII gene therapy has the potential to improve the current treatment protocols. In this report, we used a hemophilia A mouse model to compare the humoral and cellular immune responses between an E1/E2a/E3-deficient adenovirus expressing human FVIII directed by a liver-specific albumin promoter and purified recombinant FVIII protein infusion. Adenovirus-mediated FVIII expression did not elicit detectable CD4+ or CD8+ T cell responses and induced a weak antibody immune response to FVIII. In contrast, FVIII protein administration resulted in a potent anti-FVIII antibody response and moderate CD4+ T cell response. Furthermore, hemophiliac mice preimmunized with FVIII protein infusion to induce anti-FVIII immunity, and subsequently treated by adenovirus-mediated FVIII gene therapy, expressed therapeutic levels of FVIII despite the presence of low levels of anti-FVIII antibodies. No FVIII was detected in the plasma of mice with intermediate or high antibody levels, although anti-FVIII antibody levels in some vector-treated animals declined. The data support the hypothesis that liver-specific gene therapy-mediated expression of FVIII may be less immunogenic than traditional protein replacement therapy.

Topics: Adenoviridae; Albumins; Animals; Antibodies; Disease Models, Animal; Factor VIII; Gene Expression; Genetic Therapy; Genetic Vectors; Hemophilia A; Humans; Immunoglobulin Isotypes; Liver; Mice; Mice, Inbred C57BL; Organ Specificity; Ovalbumin; Promoter Regions, Genetic; T-Lymphocytes, Cytotoxic; Time Factors

In human immune deficiency virus (HIV)-seropositive hemophilia patients, a low number of CD4 + lymphocytes is found, as well as a low CD4+/CD8+ ratio. In previous studies, it has been shown that antigen-specific T-helper cell (CD4+) function was present and no excessive antigen-specific T-suppressor cell (CD8+) function could be demonstrated. In this report, we studied another activity of CD4+ cells, namely the capacity to induce T-suppressor cell activity. The results clearly show a selective dysfunction of CD4+ suppressor-inducer (Tsi) cell function. Since these HIV-seropositive hemophilia patients showed the presence of activated B cells in the peripheral circulation refractory to antigen-specific T-helper cell signals and secreting specific antibodies spontaneously, we raised the hypothesis that the activated B cells in the patients activate the Tsi cells in vivo. This constant activation leads to a functional exhaustion of the Tsi cell pool.

Topics: Antibody Formation; B-Lymphocytes; CD4-Positive T-Lymphocytes; Cells, Cultured; Hemophilia A; HIV Seropositivity; Humans; Immunity, Cellular; In Vitro Techniques; Ovalbumin; T-Lymphocytes, Regulatory

The capacity of the peripheral blood lymphocytes (PBL) to generate an antibody response in vitro T cell-dependent antigen ovalbumin was studied in 12 severe hemophilia patients who were otherwise in good health. PBL from four of 12 patients were not capable of generating such a response after stimulation in vitro, whereas all controls were normal. This negative plaque-forming cell (PFC) response coincided with the presence of antibodies directed toward human T-lymphotropic virus III/lymphadenopathy-associated virus (HTLV-III/LAV). Only one patient with antibodies against HTLV-III/LAV had a normal PFC response. The negative PFC response was not due to a deficient T helper cell activity, nor to an excessive T suppressor cell function. However, in the peripheral blood of these four patients, the presence of activated B cells that are refractory to antigen-specific T helper cell signals and secrete specific antibodies spontaneously could be demonstrated. Most of the patients showed a hyperimmunoglobulinemia. No correlation between the T4/T8 ratio and the level of the PFC response was demonstrable. From the data obtained in these investigations we raise the hypothesis that infection with HTLV-III/LAV in hemophilia patients will lead to in vivo (pre)activation of B cells that results in unresponsiveness or decreased response to antigen-specific signals.

Topics: Adolescent; Adult; Antibodies, Viral; B-Lymphocytes; Cells, Cultured; Child; Hemophilia A; Hemophilia B; HIV Antibodies; Humans; Immunoglobulin M; Immunologic Surveillance; Middle Aged; Ovalbumin; T-Lymphocytes; T-Lymphocytes, Helper-Inducer; T-Lymphocytes, Regulatory